Tumour-specific animal proteins

ABSTRACT

CASB7439 polypeptides and polynucleotides, immunogenic compositions comprising them and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing CASB7439 polypeptides and polynucleotides in diagnostics, and vaccines for prophylactic and therapeutic treatment of cancers, particularly colorectal, breast, and lung cancers, autoimmune diseases, and related conditions.

CROSS REFERENCES TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part and claims benefit toU.S. application Ser. No. 10/226,872, which was filed in the UnitedStates Patent and Trademark Office on Aug. 23, 2002, which was filed asa continuation-in-part of International Application No. PCT/EP01/01779,filed on Feb. 16, 2001, which claims priority of Great Britain PatentApplication No. 0004269.7, filed Feb. 23, 2000, which claims priority ofGreat Britain Patent Application No. 0009905.1; filed Apr. 20, 2000,which claims priority of Great Britain Patent Application No. 0021080.7,filed Aug. 25, 2000. This application also claims benefit to copendingInternational Application Serial Nos.: PCT/EP02/05011, filed 16 May 2001and PCT/EP02/01649, filed 21 Feb. 2001, the contents of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to pharmaceutical compositions andmethods for inducing an immune response against tumor-related antigens.More specifically, the invention relates to polynucleotides, hereinreferred to as CASB7439 polynucleotides, polypeptides encoded thereby(referred to herein as CASB7439 polypeptides), recombinant materials andmethods for their production and use of CASB7439 polynucleotide andpolypeptides in the diagnosis and treatment of cancer.

BACKGROUND

[0003] Polypeptides and polynucleotides of the present invention arebelieved to be important immunogens for specific prophylactic ortherapeutic immunization against tumors, because they are specificallyexpressed or highly over-expressed in tumors compared to normal cellsand can thus be targeted by antigen-specific immune mechanisms leadingto the destruction of the tumor cell. They can also be used to diagnosethe occurrence of tumor cells. Furthermore, their inappropriateexpression in certain circumstances can cause an induction ofautoimmune, inappropriate immune responses, which could be correctedthrough appropriate vaccination using the same polypeptides orpolynucleotides. In this respect the most important biologicalactivities to our purpose are the antigenic and immunogenic activitiesof the polypeptide of the present invention. A polypeptide of thepresent invention may also exhibit at least one other biologicalactivity of a CASB7439 polypeptide, which could qualify it as a targetfor therapeutic or prophylactic intervention different from that linkedto the immune response.

SUMMARY OF THE INVENTION

[0004] The present invention relates to a method of treating orpreventing cancer in a patient comprising the steps of administering atherapeutically effective amount of a polypeptide comprising an aminoacid sequence having at least 70% sequence identity to the amino acidsequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:14 over the entire lengthof SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12 or SEQ ID NO:14 respectively, wherein the polypeptide mayoptionally comprise a fusion partner or an affinity tag, whereinadministration of said polypeptide to said patient induces an immuneresponse to a tumour antigen. In another aspect, the polypeptide isadmixed with an adjuvant. In a further aspect, the tumour antigencomprises CASB7439. In another aspect, the patient has or has apotential to contract a cancer comprising colorectal, breast or lungcancer. In another aspect, the polypeptide has at least 95% sequenceidentity to SEQ ID NO:2.

[0005] In another embodiment, the present invention provides a method ofinducing an immunoresponse to CASB7439 in a human or non-human animalcomprising administering a peptide fragment of SEQ ID NO:2 to the humanor non-human animal. In one aspect, the peptide fragment is selectedfrom the group consisting of SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, and 33. In another aspect, thepeptide fragment further comprises a fusion partner. The peptidefragment or peptide fragment with fusion partner may be admixed with anadjuvant.

[0006] In another embodiment, the present invention provides a method ofmanufacturing a medicament for immunotherapeutically treating a patientsuffering from or susceptible to cancer comprising expressing a proteinin a cell comprising a polynucleotide comprising a nucleotide sequencewhich has at least 70% sequence identity to the nucleotide sequence setforth in SEQ ID NO:1 over the entire length of SEQ ID NO:1. In oneaspect, the polynucleotide has at least 95% sequence identity to SEQ IDNO:1. In another aspect, the patient is suffering from a cancercomprising colorectal, breast or lung cancer. In another aspect, thepolynucleotide is selected from the group consisting of

[0007] (a) a polynucleotide comprising a nucleotide sequence encodingSEQ ID NO:2;

[0008] (b) the coding region of polynucleotide SEQ ID NO:1; and

[0009] (c) a polynucleotide obtainable by screening an appropriatelibrary under stingent hybridization conditions with a labeled probehaving the sequence of SEQ ID NO:1 or a fragment thereof, wherein saidpolynucleotide encodes a polypeptide having simlar properties to thosefo SEQ ID NO:2.

[0010] In another embodiment, the present invention provides a method ofmanufacturing a medicament comprising a polypeptide which has at least70% sequence identity to the amino acid sequence set forth in SEQ IDNO:2 over the entire length of SEQ ID NO:2 for the manufacture of amedicament for immunotherapeutically treating a patient suffering fromor susceptible to cancer. In one aspect, the polypeptide has at least95% sequence identity to SEQ ID NO:2. In another aspect, the patient issuffering from a cancer comprising colorectal, breast or lung cancer.

[0011] In yet another embodiment, the present invention provides animmunogenic fragment of CASB7439, wherein the immunogenic fragment isimmunologically reactive with an antibody that binds to and/or a T-cellthat reacts with or binds to a polypeptide comprising SEQ ID NO:2. Inanother aspect, a pharmaceutical composition comprising such animmunogenic fragment is provided.

[0012] In yet another embodiment, the present invention providespolypeptide comprising the amino acid sequence set forth in SEQ IDNO:35. In another aspect, isolated polynucleotides encoding such apolypeptide are provided. In addition, expression vectors comprisingsuch polynucleotides are provided as well as a host cell comprising suchexpression vectors.

BRIEF DESCRIPTION OF THE FIGURES

[0013]FIG. 1: Real-time PCR data using the Taqman probe

[0014]FIG. 2: Real-time PCR expression using Sybr protocol

[0015]FIG. 3: Coomassie blue stained SDS PAGE of the cell extract fromthe strain expressing CASB7439

[0016]FIG. 4: Western blot loaded with cell extract from the strainexpressing CASB7439.

[0017]FIG. 5: Coomassie-blue stained SDS-PAGE of CASB7439 afterpurification. Lanes 1 and 5 represent the molecular weight markers;lanes 2, 3, 4 are respectively loaded with 2 μl, 4 μl and 6 μl ofpurified protein.

[0018]FIG. 6: Western blot CASB7439 after purification as revealed by ananti-polyhistidine monoclonal antibody.

[0019]FIG. 7: IHC results on colon tumour #9476 biospy.

[0020]FIG. 8: IHC results colon normal mucosa #9476 biospy.

[0021]FIG. 9: Real-time RT-PCR quantification of CASB7439 transcript ona panel of cell lines, tumor and normal tissues

[0022]FIG. 10: Real-time RT-PCR quantification (3′ UTR pimers) ofCASB7439 transcript on a panel of tumors grown in SCID mice.

[0023]FIG. 11: Real-time RT-PCR quantification (ORF primers) of CASB7439transcript on a panel of tumors grown in SCID mice

[0024]FIG. 12: Real-time RT-PCR quantification (3′ UTR pimers) ofCASB7439 transcript on an extended panel of normal and tumors tissues.

[0025]FIG. 13: Real-time RT-PCR quantification (3′ UTR pimers) ofCASB7439 transcript on an extended panel of normal and tumors tissues

[0026]FIG. 14: Real-time RT-PCR quantification (3′ UTR pimers) ofCASB7439 transcript on an extended panel of normal and tumors tissues,continued.

[0027]FIG. 15: Design of NS1-CASB7439 protein.

[0028]FIG. 16: Schematic representation of pMG1 cloning strategy(constructs 1 and 2).

[0029]FIG. 17: Coomassie blue stained SDS PAGE of the cell extract fromthe strain expressing CASB7439 construct 1.

[0030]FIG. 18: Western blot loaded with cell extract from the strainexpressing CASB7439 construct 1.

[0031]FIG. 19: Western blot CASB7439 construct 1 after purification(monoclonal anti-NS1 N terminal fragment).

[0032]FIG. 20: Comparison of Constructs 3 to 5 expression yields.Western blot on CASB7439 constructs 3 to 5 expressed in E. coli,revealed with antipeptide SB600 antibody.

[0033]FIG. 21: Expression of C7439 in HEK 293T Cells by DNA VaccineVector pJB76 (pJB16/C7439)

[0034]FIG. 22: Schematic representation of CASB7439 expressed in aadenoviral live vector

[0035]FIG. 23: Expression of C7439 in Human Fibroblasts by RecombinantAdenovirus AdC7439

[0036]FIG. 24: Real-time RT-PCR quantification of CASB7439 transcript ona population of preneoplasic lesions of lung (PLL) patients.

[0037]FIG. 25: Real-time RT-PCR quantification of CASB7439 transcript ona population oc early stage lung cancer (ESLC) patients.

[0038]FIG. 26: Real-time RT-PCR quantification of CASB7439 transcript ona population of late stage lung cancer (LSLC) patients.

[0039]FIG. 27: Real-time RT-PCR quantification of CASB7439 transcript ona panel of normal tissues.

[0040]FIG. 28: Real-time RT-PCR quantification of CASB7439 transcript ona panel of normal tissues, continued.

[0041]FIG. 29: Design of NS1-CASB7439 protein

[0042]FIG. 30: Schematic represention of pMG1 cloning strategy(constructs 1 and 2).

[0043]FIG. 31: Comparison of Constructs 3 to 5 expression yields.Western blot on CASB7439 constructs 3 to 5 expressed in E. coli,revealed with antipeptide SB600 antibody.

[0044]FIG. 32: CASB7439 peptide 1 immunization of rabbit SB598:graphical view of ELISA read-outs (Antibody titers relative to CASB7439peptide 1, straight line; antibody titers relative to KLH carrier,discontinuous line)

[0045]FIG. 33: CASB7439 peptide 1 immunization of rabbit SB599:graphical view of ELISA read-outs (Antibody titers relative to CASB7439peptide 1, straight line; antibody titers relative to KLH carrier,discontinuous line).

[0046]FIG. 34: CASB7439 peptide 2 immunization of rabbit SB600:graphical view of ELISA read-outs (Antibody titers relative to CASB7439peptide 2, straight line; antibody titers relative to KLH carrier,discontinuous line).

[0047]FIG. 35: CASB7439 peptide 2 immunization of rabbit SB601:graphical view of ELISA read-outs (Antibody titers relative to CASB7439peptide 2, straight line; antibody titers relative to KLH carrier,discontinuous line).

DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention relates to pharmaceutical compositions andmethods for inducing an immune response against tumor-related antigens.More specifically, the invention relates to polynucleotides, hereinreferred to as CASB7439 polynucleotides, polypeptides encoded thereby(referred to herein as CASB7439 polypeptides), recombinant materials andmethods for their production. In another aspect, the invention relatesto methods for using such polypeptides and polynucleotides, includingthe treatment of cancer, more particularly colorectal cancer, breastcancer, and lung cancer and autoimmune diseases and other relatedconditions. In another aspect, the invention relates to pharmaceuticalcompositions containing CASB7439 polypeptides and polynucleotides, tomethods of manufacture of such compositions and to their use inmedicine. In a further aspect, the invention relates to methods foridentifying agonists and antagonists/inhibitors using the materialsprovided by the invention, and treating conditions associated withCASB7439 polypeptide imbalance with the identified compounds. In a stillfurther aspect, the invention relates to diagnostic assays for detectingdiseases associated with inappropriate CASB7439 polypeptide activity orlevels.

[0049] In a first aspect, the present invention relates to CASB7439polypeptides. Such peptides include isolated polypeptides, comprising anamino acid sequence which has at least 70% identity, or at least 80%identity, or at least 90% identity, or at least 95% identity, or atleast 97-99% identity, to that of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:14 over the entirelength of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12 or SEQ ID NO:14 respectively. Such polypeptidesinclude those comprising the amino acid of SEQ ID NO:3, SEQ ID NO:7, SEQID NO:10 and SEQ ID NO:11.

[0050] Further peptides of the present invention include isolatedpolypeptides, in which the amino acid sequence has at least 70%identity, or at least 80% identity, or at least 90% identity, or atleast 95% identity, or at least 97-99% identity, to the amino acidsequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12 or SEQ ID NO:14 over the entire length of SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12or SEQ ID NO:14, respectively. Such polypeptides include thepolypeptides of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10 and SEQ ID NO:11.

[0051] The aforementioned polypeptides may be recombinantly produced.The polypeptides according to the invention may be purified, and aresubstantially free of any other proteins or contaminatinghost-originating material.

[0052] Further peptides of the present invention include isolatedpolypeptides encoded by a polynucleotide comprising the sequencecontained in SEQ ID NO:1.

[0053] The invention also provides an immunogenic fragment of a CASB7439polypeptide, that is a contiguous portion of the CASB7439 polypeptidewhich has the same or similar immunogenic properties to the polypeptidecomprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:7, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:14. That isto say, the fragment (if necessary when coupled to a carrier or as partof a larger fusion protein) is capable of raising an immune responsewhich recognizes the CASB7439 polypeptide. Such an immunogenic fragmentmay include, for example, the CASB7439 polypeptide lacking an N-terminalleader sequence, a transmembrane domain or a C-terminal anchor domain.The immunogenic fragment of CASB7439 according to the invention maycomprises substantially all of the extracellular domain of a polypeptidewhich has at least 70% identity, or at least 80% identity, or at least90% identity, or at least 95% identity, or at least 97-99% identity, tothat of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12 or SEQ ID NO:14 over the entire length of SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12or SEQ ID NO:14, respectively. An immunogenic fragment according to theinvention may comprise at least one epitope.

[0054] Peptide fragments incorporating an epitope of CASB7439 typicallywill comprise at least 7, or 9 or 10 contiguous amino acids from SEQ IDNO:2. Epitopes also include those shown in SEQ ID NO:16 to SEQ ID NO:33.

[0055] Peptides that incorporate these epitopes form one aspect of thepresent invention. Mimotopes which have the same characteristics asthese epitopes, and immunogens comprising such mimotopes which generatean immune response which cross-react with an epitope in the context ofthe CASB7439 molecule, also form part of the present invention.

[0056] The present invention, therefore, includes isolated peptidesencompassing these epitopes themselves, and any mimotope thereof. Themeaning of mimotope is defined as an entity which is sufficientlysimilar to the native CASB7439 epitope so as to be capable of beingrecognized by antibodies which recognize the native molecule; (Gheysen,H. M., et al., 1986, Synthetic peptides as antigens. Wiley, Chichester,Ciba foundation symposium 119, p130-149; Gheysen, H. M., 1986, MolecularImmunology, 23,7, 709-715); or are capable of raising antibodies, whencoupled to a suitable carrier, which antibodies cross-react with thenative molecule.

[0057] Peptide mimotopes of the above-identified epitopes may bedesigned for a particular purpose by addition, deletion or substitutionof elected amino acids. Thus, the peptides of the present invention maybe modified for the purposes of ease of conjugation to a proteincarrier. For example, it may be desirable for some chemical conjugationmethods to include a terminal cysteine to the epitope. In addition itmay be desirable for peptides conjugated to a protein carrier to includea hydrophobic terminus distal from the conjugated terminus of thepeptide, such that the free unconjugated end of the peptide remainsassociated with the surface of the carrier protein. This reduces theconformational degrees of freedom of the peptide, and thus increases theprobability that the peptide is presented in a conformation which mostclosely resembles that of the peptide as found in the context of thewhole molecule. For example, the peptides may be altered to have anN-terminal cysteine and a C-terminal hydrophobic amidated tail.Alternatively, the addition or substitution of a D-stereoisomer form ofone or more of the amino acids may be performed to create a beneficialderivative, for example to enhance stability of the peptide. Thoseskilled in the art will realise that such modified peptides, ormimotopes, could be a wholly or partly non-peptide mimotope wherein theconstituent residues are not necessarily confined to the 20 naturallyoccurring amino acids. In addition, these may be cyclized by techniquesknown in the art to constrain the peptide into a conformation thatclosely resembles its shape when the peptide sequence is in the contextof the whole molecule. Cyclizing a peptide may comprise the addition ofa pair of cysteine residues to allow the formation of a disulphidebridge.

[0058] Further, those skilled in the art will realise that mimotopes orimmunogens of the present invention may be larger than theabove-identified epitopes, and as such may comprise the sequencesdisclosed herein. Accordingly, the mimotopes of the present inventionmay consist of addition of N and/or C terminal extensions of a number ofother natural residues at one or both ends. The peptide mimotopes mayalso be retro sequences of the natural sequences, in that the sequenceorientation is reversed; or alternatively the sequences may be entirelyor at least in part comprised of D-stereo isomer amino acids (inversosequences). Also, the peptide sequences may be retro-inverso incharacter, in that the sequence orientation is reversed and the aminoacids are of the D-stereoisomer form. Such retro or retro-inversopeptides have the advantage of being non-self, and as such may overcomeproblems of self-tolerance in the immune system.

[0059] Alternatively, peptide mimotopes may be identified usingantibodies which are capable themselves of binding to the epitopes ofthe present invention using techniques such as phage display technology(EP 0 552 267 B1). This technique, generates a large number of peptidesequences which mimic the structure of the native peptides and are,therefore, capable of binding to anti-native peptide antibodies, but maynot necessarily themselves share significant sequence homology to thenative peptide. This approach may have significant advantages byallowing the possibility of identifying a peptide with enhancedimmunogenic properties, or may overcome any potential self-antigentolerance problems which may be associated with the use of the nativepeptide sequence. Additionally this technique allows the identificationof a recognition pattern for each native-peptide in terms of its sharedchemical properties amongst recognised mimotope sequences.

[0060] The covalent coupling of the peptide to the immunogenic carriercan be carried out in a manner well known in the art. Thus, for example,for direct covalent coupling it is possible to utilise a carbodiimide,glutaraldehyde or (N-[γ-maleimidobutyryloxy]succinimide ester, utilisingcommon commercially available heterobifunctional linkers such as CDAPand SPDP (using manufacturers instructions). After the couplingreaction, the immunogen can easily be isolated and purified by means ofa dialysis method, a gel filtration method, a fractionation method etc.

[0061] The types of carriers used in the immunogens of the presentinvention will be readily known to the man skilled in the art. Thefunction of the carrier is to provide cytokine help in order to helpinduce an immune response against the peptide. A non-exhaustive list ofcarriers which may be used in the present invention include: Keyholelimpet Haemocyanin (KLH), serum albumins such as bovine serum albumin(BSA), inactivated bacterial toxins such as tetanus or diptheria toxins(TT and DT), or recombinant fragments thereof (for example, Domain 1 ofFragment C of TT, or the translocation domain of DT), or the purifiedprotein derivative of tuberculin (PPD). Alternatively the mimotopes orepitopes may be directly conjugated to liposome carriers, which mayadditionally comprise immunogens capable of providing T-cell help. Theratio of mimotopes to carrier is in the order of 1:1 to 20:1, and eachcarrier may carry between 3-15 peptides.

[0062] In one embodiment of the invention a carrier is Protein D fromHaemophilus influenzae (EP 0 594 610 B1). Protein D is an IgD-bindingprotein from Haemophilus influenzae and has been patented by Forsgren(WO 91/18926, granted EP 0 594 610 B1). In some circumstances, forexample in recombinant immunogen expression systems it may be desirableto use fragments of protein D, for example Protein D ⅓^(rd) (comprisingthe N-terminal 100-110 amino acids of protein D (GB 9717953.5)).

[0063] Another method of presenting the peptides of the presentinvention is in the context of a recombinant fusion molecule. Forexample, EP 0 421 635 B describes the use of chimaeric hepadnavirus coreantigen particles to present foreign peptide sequences in a virus-likeparticle. As such, immunogens of the present invention may comprisepeptides presented in chimaeric particles consisting of hepatitis B coreantigen. Additionally, the recombinant fusion proteins may comprise themimotopes of the present invention and a carrier protein, such as NS1 ofthe influenza virus. For any recombinantly expressed protein which formspart of the present invention, the nucleic acid which encodes saidimmunogen also forms an aspect of the present invention.

[0064] Peptides used in the present invention can be readily synthesisedby solid phase procedures well known in the art. Suitable syntheses maybe performed by utilising “T-boc” or “F-moc” procedures. Cyclic peptidescan be synthesised by the solid phase procedure employing the well-known“F-moc” procedure and polyamide resin in the fully automated apparatus.Alternatively, those skilled in the art will know the necessarylaboratory procedures to perform the process manually. Techniques andprocedures for solid phase synthesis are described in ‘Solid PhasePeptide Synthesis: A Practical Approach’ by E. Atherton and R. C.Sheppard, published by IRL at Oxford University Press (1989).Alternatively, the peptides may be produced by recombinant methods,including expressing nucleic acid molecules encoding the mimotopes in abacterial or mammalian cell line, followed by purification of theexpressed mimotope. Techniques for recombinant expression of peptidesand proteins are known in the art, and are described in Maniatis, T.,Fritsch, E. F. and Sambrook et al., Molecular cloning, a laboratorymanual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989).

[0065] In a further embodiment of the invention is provided a method ofproducing a polypeptide as described herein. The process of theinvention may be performed by conventional recombinant techniques suchas described in Maniatis et al., Molecular Cloning—A Laboratory Manual;Cold Spring Harbor, 1982-1989. Accordingly there is provided a processfor producing a polypeptide according to the invention, comprisingculturing a host cell under conditions sufficient for the production ofsaid polypeptide and recovering the polypeptide from the culture medium.In particular, the process of the invention may comprise the steps of:

[0066] i) preparing a replicable or integrating expression vectorcapable, in a host cell, of expressing a DNA polymer comprising anucleotide sequence that encodes the protein or an immunogenicderivative thereof;

[0067] ii) transforming a host cell with said vector;

[0068] ii) culturing said transformed host cell under conditionspermitting expression of said DNA polymer to produce said protein; and

[0069] iv) recovering said protein.

[0070] The polypeptides or immunogenic fragment of the invention may bein the form of the “mature” protein or may be a part of a larger proteinsuch as a precursor or a fusion protein. It is often advantageous toinclude an additional amino acid sequence which contains secretory orleader sequences, pro-sequences, sequences which aid in purificationsuch as multiple histidine residues, or an additional sequence forstability during recombinant production. Furthermore, addition ofexogenous polypeptide or lipid tail or polynucleotide sequences toincrease the immunogenic potential of the final molecule is alsoconsidered.

[0071] In one aspect, the invention relates to genetically engineeredsoluble fusion proteins comprising a polypeptide of the presentinvention, or a fragment thereof, and various portions of the constantregions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). An immunoglobulin may include theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa. Furthermore, thisinvention relates to processes for the preparation of these fusionproteins by genetic engineering, and to the use thereof for drugscreening, diagnosis and therapy. Another aspect of the inventionrelates to the use of a polypeptide or a polynucleotide in themanufacture of a vaccine for immunotherapeutically treating a patientsuffering from or susceptible to carcinoma, especially colon cancer orother colon-associated tumours or diseases. A further aspect of theinvention also relates to polynucleotides encoding such fusion proteins.Examples of fusion protein technology can be found in InternationalPatent Application Nos. WO94/29458 and WO94/22914.

[0072] The proteins may be chemically conjugated, or expressed asrecombinant fusion proteins allowing increased levels to be produced inan expression system as compared to non-fused protein. The fusionpartner may assist in providing T helper epitopes (immunological fusionpartner), including T helper epitopes recognised by humans, or assist inexpressing the protein (expression enhancer) at higher yields than thenative recombinant protein. The fusion partner may be both animmunological fusion partner and expression enhancing partner.

[0073] Fusion partners include protein D from Haemophilus influenza Band the non-structural protein from influenzae virus, NS1(hemagglutinin). Another immunological fusion partner is the proteinknown as LYTA. The C terminal portion of the molecule may be used. Lytais derived from Streptococcus pneumoniae which synthesize anN-acetyl-L-alanine amidase, amidase LYTA, (coded by the lytA gene {Gene,43 (1986) page 265-272} an autolysin that specifically degrades certainbonds in the peptidoglycan backbone. The C-terminal domain of the LYTAprotein is responsible for the affinity to the choline or to somecholine analogues such as DEAE. This property has been exploited for thedevelopment of E. coli C-LYTA expressing plasmids useful for expressionof fusion proteins. Purification of hybrid proteins containing theC-LYTA fragment at its amino terminus has been described {Biotechnology:10, (1992) page 795-798}. It is possible to use the repeat portion ofthe Lyta molecule found in the C terminal end starting at residue 178,for example residues 188-305.

[0074] The present invention also includes xenogeneic forms (also termedortholog forms) of the aforementioned polypeptides, said xenogeneicforms referring to an antigen having substantial sequence identity tothe human antigen (also termed autologous antigen) which serves as areference antigen but which is derived from a different non-humanspecies. In this context the substantial identity refers to concordanceof an amino acid sequence with another amino acid sequence or of apolynucleotide sequence with another polynucleotide sequence when suchsequence are arranged in a best fit alignment in any of a number ofsequence alignment proteins known in the art. By substantial identity ismeant at least 70-95%, or at least 85-95%, or at least 90%-95%, sequenceidentity between the compared sequences. Therefore according to theinvention the xenogeneic CASB7439 polypeptide will be a CASB7439polypeptide which is xenogeneic with respect to human CASB7439, in otherwords which is isolated from a species other than human. In oneembodiment, the polypeptide is isolated from mouse, rat, pig, or rhesusmonkey. Accordingly the present invention also provides a method ofinducing an immune response against human CASB7439 having an amino acidsequence as set forth in any of the sequences SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:7, SEQ ID NO:10 or SEQ ID NO:11 in a human, comprisingadministering to the subject an effective dosage of a compositioncomprising a xenogeneic form of said human CASB7439 as described herein.Another embodiment is a method of inducing an immune response againsthuman CASB7439 using the xenogeneic CASB7439 isolated from mouse, rat,pig or rhesus monkey. Another method of inducing an immune responseaccording to the present invention includes using an antigen compositionincluding a live viral expression system which expresses said xenogeneicantigen. The xenogeneic CASB7439 polypeptide may have the sequence setforth in SEQ ID No12 (mouse) or in SEQ ID No14 (rat).

[0075] The isolated xenogeneic CASB7439 polypeptide will generally sharesubstantial sequence similarity, and include isolated polypeptidescomprising an amino acid sequence which has at least 70% identity, or atleast 80% identity, or at least 90% identity, or at least 95% identity,or at least 97-99% identity, to that of SEQ ID NO:12 or SEQ ID No14 overthe entire length of SEQ ID NO:12 or SEQ ID No14. Accordingly thexenogeneic polypeptide may comprise an immunogenic fragment of thepolypeptide of SEQ ID NO:12 or SEQ ID NO:14 in which the immunogenicactivity of the immunogenic fragment is substantially the same as thepolypeptide of SEQ ID NO:12 or SEQ ID NO:14. In addition the xenogeneicCASB7439 polypeptide can be a fragment of at least about 20 consecutiveamino acids, or about 30, or about 50, or about 100, or 150 contiguousamino acids selected from the amino acid sequences as shown in SEQ IDNO:12 or in SEQ ID No14. More particularly xenogeneic CASB7439 fragmentswill retain some functional property, which may be immunologicalactivity, of the larger molecule set forth in SEQ ID NO:12 or in SEQ IDNo14, and are useful in the methods described herein (e.g. inpharmaceutical and vaccine compositions, in diagnostics, etc.). Inparticular the fragments will be able to generate an immune responseagainst the human counterpart, such as the generation of cross-reactiveantibodies which react with the autologous human form of CASB7439 as setforth in any of the SEQ ID NO:2. In a specific embodiment, thexenogeneic polypeptide of the invention may be part of a larger fusion,comprising the xenogeneic CASB7439 polypeptide or fragment thereof and aheterologous protein or part of a protein acting as a fusion partner asdescribed hereabove.

[0076] The present invention also includes variants of theaforementioned polypeptides, that is polypeptides that vary from thereferents by conservative amino acid substitutions, whereby a residue issubstituted by another with like characteristics. Typical suchsubstitutions are among Ala, Val, Leu and Ile; among Ser and Thr; amongthe acidic residues Asp and Glu; among Asn and Gln; and among the basicresidues Lys and Arg; or aromatic residues Phe and Tyr. Variants inwhich several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted,deleted, or added in any combination are also included within theinvention.

[0077] Polypeptides of the present invention can be prepared in anysuitable manner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

[0078] In a further aspect, the present invention relates to CASB7439polynucleotides. Such polynucleotides include isolated polynucleotidescomprising a nucleotide sequence encoding a polypeptide which has atleast 70% identity, or at least 80% identity, or at least 90% identity,or at least 95% identity, to the amino acid sequence of SEQ ID NO:2, SEQID NO:3, SEQ ID NO:7, SEQ ID NO:10 or SEQ ID NO:11, over the entirelength of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10 or SEQ IDNO:11 respectively. In this regard, encoded polypeptides which have atleast 97% or at least 98-99% identity or at least 99% identity areconsidered part of the invention.

[0079] Further polynucleotides of the present invention include isolatedpolynucleotides comprising a nucleotide sequence that has at least 70%identity, or at least 80% identity, or at least 90% identity, or atleast 95% identity, to a nucleotide sequence encoding a polypeptide ofSEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, or SEQ ID NO:11,over the entire coding region. In this regard, polynucleotides whichhave at least 97% or at least 98-99% identity or at least 99% identityare also included.

[0080] Further polynucleotides of the present invention include isolatedpolynucleotides comprising a nucleotide sequence which has at least 70%identity, or at least 80% identity, or at least 90% identity, or atleast 95% identity, to SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:8 or SEQ ID NO:9, over the entire length of saidsequences, or to the coding sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:9 over the entire length ofsaid coding sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:8 or SEQ ID NO:9. In this regard, polynucleotides whichhave at least 97% identity or at least 98-99% identity or at least 99%identity are also included within the invention. Such polynucleotidesinclude a polynucleotide comprising the polynucleotide of SEQ ID NO:1,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:9 aswell as the polynucleotide of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:9 or the coding region of SEQ ID NO:1,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:9.

[0081] The present invention also provides a nucleic acid encoding theaforementioned xenogeneic proteins of the present invention and theiruse in medicine. In another embodiment, the xenogeneic CASB7439polynucleotide for use in pharmaceutical compositions has the sequenceset forth in SEQ ID No13 (mouse) or in SEQ ID No15 (rat). The isolatedxenogeneic CASB7439 polynucleotides according to the invention may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide of the present invention. In other related embodiments,the present invention provides polynucleotide variants havingsubstantial identity to the sequences disclosed herein in SEQ ID No13 orin SEQ ID No15, for example those comprising at least 70% sequenceidentity, or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% orhigher, sequence identity compared to a polynucleotide sequence of thisinvention using the methods described herein, (e.g., BLAST analysisusing standard parameters). In a related embodiment, the isolatedxenogeneic polynucleotide of the invention will comprise a nucleotidesequence encoding a polypeptide that has at least 90%, or 95% and above,identity to the amino acid sequence of SEQ ID NO:12 or of SEQ ID NO:14,over the entire length of SEQ ID NO:12 or of SEQ ID NO:14; or anucleotide sequence complementary to said isolated polynucleotide.

[0082] The invention also provides polynucleotides which arecomplementary to all the above described polynucleotides.

[0083] Said polynucleotides can be inserted in a suitable plasmid,recombinant microorganism vector or a recombinant live microorganism andused for immunization (see for example Wolff et. al., Science247:1465-1468 (1990); Corr et. al., J. Exp. Med. 184:1555-1560 (1996);Doe et. al., Proc. Natl. Acad. Sci. 93:8578-8583 (1996)). Accordinglythere is provided in the present invention an expression vector orrecombinant live microorganism comprising said polynucleotides ashereabove defined.

[0084] The invention also provides a fragment of a CASB7439polynucleotide which when administered to a subject has the sameimmunogenic properties as the polynucleotide of SEQ ID NO:1, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13or SEQ ID NO:15.

[0085] The invention also provides a polynucleotide encoding animmunological fragment of a CASB7439 polypeptide as hereinbeforedefined.

[0086] The fragments have a level of immunogenic activity of at leastabout 50%, or at least about 70% or at least about 90% of the level ofimmunogenic activity of a polypeptide sequence set forth in SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10 or SEQ ID NO:11, SEQ ID NO:12 orSEQ ID NO:14 or a polypeptide sequence encoded by a polynucleotidesequence set forth in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13 or SEQ ID NO:15.

[0087] The polypeptide fragments according to the invention may compriseat least about 5, 10, 15, 20, 25, 50, or 100 contiguous amino acids, ormore, including all intermediate lengths, of a polypeptide compositionset forth herein, such as those set forth in SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:14,or those encoded by a polynucleotide sequence set forth in a sequence ofSEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:13 or SEQ ID NO:15.

[0088] The nucleotide sequence of SEQ ID NO:1 is a cDNA sequence whichcomprises a polypeptide encoding sequence (nucleotide 545 to 1126)encoding a polypeptide of 193 amino acids, the polypeptide of SEQ IDNO:2. The nucleotide sequence encoding the polypeptide of SEQ ID NO:2may be identical to the polypeptide encoding sequence contained in SEQID NO:1 or it may be a sequence other than the one contained in SEQ IDNO:1, which, as a result of the redundancy (degeneracy) of the geneticcode, also encodes the polypeptide of SEQ ID NO:2. The polypeptide ofthe SEQ ID NO:2 is structurally related to other proteins of the achaetescute family, and is also named “human Achaete Scute homologue 2”(HASH2) (accession number NP_(—)005161 and AAB86993).

[0089] Human Achaete Scute homologue 2 (HASH2) gene, officiallydesignated human ASCL2 (Achaete Scute complex like 2) is a homologue ofthe Drosophila Achaete and Scute genes. Human ASCL2 is expressed in theextravillus trophoblasts of the developing placenta only, and maps onchromosome 11p15 close to IGF2 and H19. The mouse achaete-scutehomolog-2 gene (MASH2) encodes a transcription factor playing a role inthe development of the trophoblast. The Mash2 gene is paternallyimprinted in the mouse, and the lack of human ASCL2 expression innon-malignant hydatidiform (androgenetic) moles indicates that humanAsc12 is also imprinted in man.

[0090] Asc12 genes are members of the basic helix-loop-helix (BHLH)family of transcription factors. They activate transcription by bindingto the E box (5′-CANNTG-3′). Dimerization with other BHLH proteins isrequired for efficient DNA binding. They are involved in thedetermination of the neuronal precursors in the peripheral nervoussystem and the central nervous system in drosophila melanogaster, andprobably in mammals as well.

[0091] The complementary strand of the nucleotide sequence of SEQ IDNO:1 is the polynucleotide sequence of SEQ ID NO:6. This strand alsocomprises two other polypeptide encoding sequences. The firstpolypeptide encoding sequence (nucleotide 1184 to 399 of SEQ ID:1,nucleotide 608 to 1393 of SEQ ID NO:6) encodes a polypeptide of 262amino acids, the polypeptide of SEQ ID NO:3. The second polypeptideencoding sequence (nucleotide 840 to 262 of SEQ ID NO:1, nucleotide 952to 1530 of SEQ ID NO:6) encodes a polypeptide of 193 amino acids, thepolypeptide of SEQ ID NO: 11. The nucleotide sequence encoding thepolypeptides of SEQ ID NO:3 and SEQ ID NO:11 may be identical to thepolypeptides encoding sequence contained in SEQ ID NO:6 or it may be asequence other than the one contained in SEQ ID NO: 6, which, as aresult of the redundancy (degeneracy) of the genetic code, also encodesthe polypeptides of SEQ ID NO:3 and 11. The polypeptide of the SEQ IDNO:3 is structurally related to other proteins of the splicingcoactivator protein family, having homology and/or structural similaritywith homo sapiens splicing coactivator subunit srm300 (genbank accessionAAF21439). The polypeptide of SEQ ID NO:11 is not related to any knownprotein. Polypeptide sequences as set forth in SEQ ID NO:3 and SEQ IDNO:11, and polynucleotide sequences as set forth in SEQ ID NO:6 arenovel and also form part of the invention.

[0092] Polypeptides and polynucleotides of the present invention areexpected to have, inter alia, similar biological functions/properties totheir homologous polypeptides and polynucleotides. Furthermore,polypeptides, immunological fragments and polynucleotides of the presentinvention have at least one activity of either SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3 or SEQ ID NO:11 as appropriate.

[0093] The present invention also relates to partial or other incompletepolynucleotide and polypeptide sequences which were first identifiedprior to the determination of the corresponding full length sequences ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:11.

[0094] Accordingly, in a further aspect, the present invention providesfor an isolated polynucleotide which:

[0095] (a) comprises a nucleotide sequence which has at least 70%identity, or 80% identity, or at least 90% identity, or at least 95%identity, or at least 97-99% identity to SEQ ID NO:4 and 5 over theentire length of SEQ ID NO 4 and 5;

[0096] (b) has a nucleotide sequence which has at least 70% identity, orat least 80% identity, or at least 90% identity, or at least 95%identity, or at least 97-99% identity, to SEQ ID NO:1 or SEQ ID NO:6over the entire length of SEQ ID NO:4 and SEQ ID NO:5 respectively;

[0097] (c) the polynucleotide of SEQ ID NO:4 and SEQ ID NO:5; or

[0098] (d) a nucleotide sequence encoding a polypeptide which has atleast 70% identity, or at least 80% identity, or at least 90% identity,or at least 95% identity, or at least 97-99% identity, to the amino acidsequence of SEQ ID NO:2 and SEQ ID NO:7 respectively, over the entirelength of SEQ ID NO:2 and 7, as well as the polynucleotides of SEQ IDNO:4 and 5.

[0099] The present invention further provides for a polypeptide which:

[0100] (a) comprises an amino acid sequence which has at least 70%identity, or at least 80% identity, or at least 90% identity, or atleast 95% identity, or at least 97-99% identity, to that of SEQ ID NO:2and 7 over the entire length of SEQ ID NO:2 or 7;

[0101] (b) has an amino acid sequence which is at least 70% identity, orat least 80% identity, or at least 90% identity, or at least 95%identity, or at least 97-99% identity, to the amino acid sequence of SEQID NO:2 or 7 over the entire length of SEQ ID NO:2 or 7;

[0102] (c) comprises the amino acid of SEQ ID NO:2 or 7; and

[0103] (d) is the polypeptide of SEQ ID NO: 7;

[0104] as well as polypeptides encoded by a polynucleotide comprisingthe sequence contained in SEQ ID NO:4 and 5.

[0105] Polynucleotides of the present invention may be obtained, usingstandard cloning and screening techniques, from a cDNA library derivedfrom mRNA in cells of human colon cancer, (for example Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring harborLaboratory Press, Cold Spring harbor, N.Y. (1989)). Polynucleotides ofthe invention can also be obtained from natural sources such as genomicDNA libraries or can be synthesized using well known and commerciallyavailable techniques.

[0106] When polynucleotides of the present invention are used for therecombinant production of polypeptides of the present invention, thepolynucleotide may include the coding sequence for the maturepolypeptide, by itself; or the coding sequence for the maturepolypeptide in reading frame with other coding sequences, such as thoseencoding a leader or secretory sequence, a pre-, or pro- orprepro-protein sequence, or other fusion peptide portions. For example,a marker sequence which facilitates purification of the fusedpolypeptide can be encoded. In certain embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., ProcNatl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotidemay also contain non-coding 5′ and 3′ sequences, such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

[0107] Further embodiments of the present invention includepolynucleotides encoding polypeptide variants which comprise the aminoacid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11,SEQ ID NO:13 or SEQ ID NO:15 and in which several, for instance from 5to 10, 1 to 5, 1 to 3, 1 to 2 or 1, amino acid residues are substituted,deleted or added, in any combination.

[0108] Polynucleotides which are identical or sufficiently identical toa nucleotide sequence contained in SEQ ID NO:1 or in SEQ ID NO:6, may beused as hybridization probes for cDNA and genomic DNA or as primers fora nucleic acid amplification (PCR) reaction, to isolate full-lengthcDNAs and genomic clones encoding polypeptides of the present inventionand to isolate cDNA and genomic clones of other genes (including genesencoding paralogs from human sources and orthologs and paralogs fromspecies other than human) that have a high sequence similarity to SEQ IDNO:1 or to SEQ ID NO:6. Typically these nucleotide sequences are 70%identical, 80% identical, 90% identical, or 95% identical to that of thereferent. The probes or primers will generally comprise at least 15nucleotides, or at least 30 nucleotides and may have at least 50nucleotides. Particularly, probes may have between 30 and 50nucleotides. Particularly primers may have between 20 and 25nucleotides. In particular, polypeptides or polynucleotides derived fromsequences from homologous animal origin could be used as immunogens toobtain a cross-reactive immune response to the human gene.

[0109] A polynucleotide encoding a polypeptide of the present invention,including homologs from species other than human, may be obtained by aprocess which comprises the steps of screening an appropriate libraryunder stringent hybridization conditions with a labeled probe having thesequence of SEQ ID NO:1 or SEQ ID NO:6 or a fragment thereof; andisolating full-length cDNA and genomic clones containing saidpolynucleotide sequence. Such hybridization techniques are well known tothe skilled artisan. Stringent hybridization conditions may includeovernight incubation at 42° C. in a solution comprising: 50% formamide,5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate(pH7.6), 5× Denhardt's solution, 10 % dextran sulfate, and 20microgram/ml denatured, sheared salmon sperm DNA; followed by washingthe filters in 0.1×SSC at about 65° C. Thus the present invention alsoincludes polynucleotides obtainable by screening an appropriate libraryunder stringent hybridization conditions with a labeled probe having thesequence of SEQ ID NO:1 or SEQ ID NO:6 or a fragment thereof.

[0110] The skilled artisan will appreciate that, in many cases, anisolated cDNA sequence will be incomplete, in that the region coding forthe polypeptide is short at the 5′ end of the cDNA.

[0111] There are several methods available and well known to thoseskilled in the art to obtain full-length cDNAs, or extend short cDNAs,for example those based on the method of Rapid Amplification of cDNAends (RACE) (see, for example, Frohman et al., PNAS USA 85, 8998-9002,1988). Recent modifications of the technique, exemplified by theMarathon™ technology (Clontech Laboratories Inc.) for example, havesignificantly simplified the search for longer cDNAs. In the Marathon™technology, cDNAs have been prepared from mRNA extracted from a chosentissue and an ‘adaptor’ sequence ligated onto each end. Nucleic acidamplification (PCR) is then carried out to amplify the ‘missing’ 5° endof the cDNA using a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using‘nested’ primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3′ in the adaptor sequence and a gene specific primer thatanneals further 5′ in the known gene sequence). The products of thisreaction can then be analysed by DNA sequencing and a full-length cDNAconstructed either by joining the product directly to the existing cDNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5′ primer.

[0112] Recombinant polypeptides of the present invention may be preparedby processes well known in the art from genetically engineered hostcells comprising expression systems. Accordingly, in a further aspect,the present invention relates to an expression system which comprises apolynucleotide of the present invention, to host cells which aregenetically engineered with such expression sytems and to the productionof polypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.

[0113] For recombinant production, host cells can be geneticallyengineered to incorporate expression systems or portions thereof forpolynucleotides of the present invention. Introduction ofpolynucleotides into host cells can be effected by methods described inmany standard laboratory manuals, such as Davis et al., Basic Methods inMolecular Biology (1986) and Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989). Such methods include, for instance, calciumphosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introduction orinfection.

[0114] The proteins of the invention may be coexpressed with thioredoxinin trans (TIT). Coexpression of thioredoxin in trans versus in cis keepsantigen free of thioredoxin without the need for protease. Thioredoxincoexpression eases the solubilisation of the proteins of the invention.Thioredoxin coexpression has also a significant impact on proteinpurification yield, on purified-protein solubility and quality.

[0115] Representative examples of appropriate hosts include bacterialcells, such as Streptococci, Staphylococci, E. coli, Streptomyces andBacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 andBowes melanoma cells; and plant cells.

[0116] A great variety of expression systems can be used, for instance,chromosomal, episomal and virus-derived systems, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression systems may containcontrol regions that regulate as well as engender expression. Generally,any system or vector which is able to maintain, propagate or express apolynucleotide to produce a polypeptide in a host may be used. Theappropriate nucleotide sequence may be inserted into an expressionsystem by any of a variety of well-known and routine techniques, suchas, for example, those set forth in Sambrook et al., Molecular Cloning,A Laboratory Manual (supra). Appropriate secretion signals may beincorporated into the desired polypeptide to allow secretion of thetranslated protein into the lumen of the endoplasmic reticulum, theperiplasmic space or the extracellular environment. These signals may beendogenous to the polypeptide or they may be heterologous signals.

[0117] The expression system may also be a recombinant livemicroorganism, such as a virus or bacterium. The gene of interest can beinserted into the genome of a live recombinant virus or bacterium.Inoculation and in vivo infection with this live vector will lead to invivo expression of the antigen and induction of immune responses.

[0118] Therefore, in certain embodiments, polynucleotides encodingimmunogenic polypeptides of the present invention are introduced intosuitable mammalian host cells for expression using any of a number ofknown viral-based systems. In one illustrative embodiment, retrovirusesprovide a convenient and effective platform for gene delivery systems. Aselected nucleotide sequence encoding a polypeptide of the presentinvention can be inserted into a vector and packaged in retroviralparticles using techniques known in the art. The recombinant virus canthen be isolated and delivered to a subject. A number of illustrativeretroviral systems have been described (e.g., U.S. Pat. No. 5,219,740;Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990)Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852;Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; andBoris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.

[0119] In addition, a number of illustrative adenovirus-based systemshave also been described. Unlike retroviruses which integrate into thehost genome, adenoviruses persist extrachromosomally thus minimizing therisks associated with insertional mutagenesis (Haj-Ahmad and Graham(1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921;Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al.(1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58;Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993)Human Gene Therapy 4:461-476).

[0120] Various adeno-associated virus (AAV) vector systems have alsobeen developed for polynucleotide delivery. AAV vectors can be readilyconstructed using techniques well known in the art. See, e.g., U.S. Pat.Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shellingand Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.Med. 179:1867-1875.

[0121] Additional viral vectors useful for delivering the nucleic acidmolecules encoding polypeptides of the present invention by genetransfer include those derived from the pox family of viruses, such asvaccinia virus and avian poxvirus. By way of example, vaccinia virusrecombinants expressing the novel molecules can be constructed asfollows. The DNA encoding a polypeptide is first inserted into anappropriate vector so that it is adjacent to a vaccinia promoter andflanking vaccinia DNA sequences, such as the sequence encoding thymidinekinase (TK). This vector is then used to transfect cells which aresimultaneously infected with vaccinia. Homologous recombination servesto insert the vaccinia promoter plus the gene encoding the polypeptideof interest into the viral genome. The resulting TK.sup.(−) recombinantcan be selected by culturing the cells in the presence of5-bromodeoxyuridine and picking viral plaques resistant thereto.

[0122] A vaccinia-based infection/transfection system can beconveniently used to provide for inducible, transient expression orcoexpression of one or more polypeptides described herein in host cellsof an organism. In this particular system, cells are first infected invitro with a vaccinia virus recombinant that encodes the bacteriophageT7 RNA polymerase. This polymerase displays exquisite specificity inthat it only transcribes templates bearing T7 promoters. Followinginfection, cells are transfected with the polynucleotide orpolynucleotides of interest, driven by a T7 promoter. The polymeraseexpressed in the cytoplasm from the vaccinia virus recombinanttranscribes the transfected DNA into RNA which is then translated intopolypeptide by the host translational machinery. The method provides forhigh level, transient, cytoplasmic production of large quantities of RNAand its translation products. See, e.g., Elroy-Stein and Moss, Proc.Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al. Proc. Natl.Acad. Sci. USA (1986) 83:8122-8126.

[0123] Alternatively, avipoxviruses, such as the fowlpox and canarypoxviruses, can also be used to deliver the coding sequences of interest.Recombinant avipox viruses, expressing immunogens from mammalianpathogens, are known to confer protective immunity when administered tonon-avian species. The use of an Avipox vector is particularly desirablein human and other mammalian species since members of the Avipox genuscan only productively replicate in susceptible avian species andtherefore are not infective in mammalian cells. Methods for producingrecombinant Avipoxviruses are known in the art and employ geneticrecombination, as described above with respect to the production ofvaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

[0124] Any of a number of alphavirus vectors can also be used fordelivery of polynucleotide compositions of the present invention, suchas those vectors described in U.S. Pat. Nos. 5,843,723; 6,015,686;6,008,035 and 6,015,694. Certain vectors based on Venezuelan EquineEncephalitis (VEE) can also be used, illustrative examples of which canbe found in U.S. Pat. Nos. 5,505,947 and 5,643,576.

[0125] Moreover, molecular conjugate vectors, such as the adenoviruschimeric vectors described in Michael et al. J. Biol. Chem. (1993)268:6866-6869 and Wagner et al. Proc. Natl. Acad Sci. USA (1992)89:6099-6103, can also be used for gene delivery under the invention.

[0126] Additional illustrative information on these and other knownviral-based delivery systems can be found, for example, in Fisher-Hochet al., Proc. Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al.,Ann. N.Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21,1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973;U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805;Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science252:431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219,1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502,1993; Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al.,Cir. Res. 73:1202-1207, 1993.

[0127] The recombinant live microorganisms described above can bevirulent, or attenuated in various ways in order to obtain livevaccines. Such live vaccines also form part of the invention.

[0128] In certain embodiments, a polynucleotide may be integrated intothe genome of a target cell. This integration may be in the specificlocation and orientation via homologous recombination (gene replacement)or it may be integrated in a random, non-specific location (geneaugmentation). In yet further embodiments, the polynucleotide may bestably maintained in the cell as a separate, episomal segment of DNA.Such polynucleotide segments or “episomes” encode sequences sufficientto permit maintenance and replication independent of or insynchronization with the host cell cycle. The manner in which theexpression construct is delivered to a cell and where in the cell thepolynucleotide remains is dependent on the type of expression constructemployed.

[0129] In another embodiment of the invention, a polynucleotide isadministered/delivered as “naked” DNA, for example as described in Ulmeret al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells.

[0130] In still another embodiment, a composition of the presentinvention can be delivered via a particle bombardment approach, many ofwhich have been described. In one illustrative example, gas-drivenparticle acceleration can be achieved with devices such as thosemanufactured by Powderject Pharmaceuticals PLC (Oxford, UK) andPowderject Vaccines Inc. (Madison, Wis.), some examples of which aredescribed in U.S. Pat. Nos. 5,846,796; 6,010,478; 5,865,796; 5,584,807;and EP Patent No. 0500 799. This approach offers a needle-free deliveryapproach wherein a dry powder formulation of microscopic particles, suchas polynucleotide or polypeptide particles, are accelerated to highspeed within a helium gas jet generated by a hand held device,propelling the particles into a target tissue of interest.

[0131] In a related embodiment, other devices and methods that may beuseful for gas-driven needle-less injection of compositions of thepresent invention include those provided by Bioject, Inc. (Portland,Oreg.), some examples of which are described in U.S. Pat. Nos.4,790,824; 5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and5,993,412.

[0132] Polypeptides of the present invention can be recovered andpurified from recombinant cell cultures by well-known methods includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. Ion metalaffinity chromatography (IMAC) may be employed for purification. Wellknown techniques for refolding proteins may be employed to regenerateactive conformation when the polypeptide is denatured duringintracellular synthesis, isolation and or purification.

[0133] Another important aspect of the invention relates to a method forinducing, re-inforcing or modulating an immunological response in amammal which comprises inoculating the mammal with a fragment or theentire polypeptide or polynucleotide of the invention, adequate toproduce antibody and/or T cell immune response for immunoprophylaxis orfor therapeutic treatment of cancer, more particularly colorectalcancer, and autoimmune disease and related conditions. Yet anotheraspect of the invention relates to a method of inducing, re-inforcing ormodulating immunological response in a mammal which comprises,delivering a polypeptide of the present invention via a vector or celldirecting expression of the polynucleotide and coding for thepolypeptide in vivo in order to induce such an immunological response toproduce immune responses for prophylaxis or treatment of said mammalfrom diseases.

[0134] A further aspect of the invention relates to animmunological/vaccine formulation (composition) and to their use inmedicine. These compositions, when introduced into a mammalian host,induce, re-inforce or modulate an immunological response in that mammalto a polypeptide of the present invention wherein the compositioncomprises a polypeptide or polynucleotide of the invention or animmunological fragment thereof as herein before defined. Moreparticularly the immunogenic composition according to the presentinvention comprises a safe and effective amount of a CASB7439polypeptide, or immunogenic fragment thereof wherein the CASB7439polypeptide is selected from the group comprising SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ IDNO:14. In another embodiment, the imunogenic composition comprises asafe and effective amount of a CASB7439-encoding polynucleotide, orfragment thereof wherein the CASB7439-encoding polynucleotide isselected from the group comprising SEQ ID NO:1, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13 or SEQ IDNO:15.

[0135] The vaccine formulation according to the invention may furthercomprise a suitable, i.e. pharmaceutically acceptable carrier. Since apolypeptide may be broken down in the stomach, it may be administeredparenterally (for instance, subcutaneous, intramuscular, intravenous, orintradermal injection). Formulations suitable for parenteraladministration include aqueous and non-aqueous sterile injectionsolutions which may contain anti-oxidants, buffers, bacteriostats andsolutes which render the formulation isotonic with the blood of therecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use.

[0136] A further aspect of the invention relates to the in vitroinduction of immune responses to a fragment or the entire polypeptide orpolynucleotide of the present invention or a molecule comprising thepolypeptide or polynucleotide of the present invention, using cells fromthe immune system of a mammal, and reinfusing these activated immunecells of the mammal for the treatment of disease. Activation of thecells from the immune system is achieved by in vitro incubation with theentire polypeptide or polynucleotide of the present invention or amolecule comprising the polypeptide or polynucleotide of the presentinvention in the presence or absence of various immunomodulatormolecules.

[0137] A further aspect of the invention relates to the immunization ofa mammal by administration of antigen presenting cells modified by invitro loading with part or the entire polypeptide of the presentinvention or a molecule comprising the polypeptide of the presentinvention and administered in vivo in an immunogenic way. Alternatively,antigen presenting cells can be transfected in vitro with a vectorcontaining a fragment or the entire polynucleotide of the presentinvention or a molecule comprising the polynucleotide of the presentinvention, such as to express the corresponding polypeptide, andadministered in vivo in an immunogenic way. Accordingly, thepharmaceutical compositions of the invention will comprise an effectiveamount of antigen presenting cells, modified by in vitro loading with aCASB7439 polypeptide, or genetically modified in vitro to express aCASB7439 polypeptide and a pharmaceutically effective carrier.

[0138] According to another embodiment, the pharmaceutical/immunogeniccompositions described herein will comprise one or more immunostimulantsin addition to the immunogenic polynucleotide, polypeptide, antibody,T-cell and/or antigen presenting cell (APC) compositions of thisinvention. Accordingly there is herein provided a process for theproduction of said immunogenic composition, comprising admixing aCASB7439 polypeptide or a CASB7439-encoding polynucleotide with asuitable adjuvant/immunostimulant, diluent or other pharmaceuticallyacceptable carrier. An immunostimulant refers to essentially anysubstance that enhances or potentiates an immune response (antibodyand/or cell-mediated) to an exogenous antigen. One type ofimmunostimulant comprises an adjuvant. Many adjuvants contain asubstance designed to protect the antigen from rapid catabolism, such asaluminum hydroxide or mineral oil, and a stimulator of immune responses,such as lipid A, Bortadella pertussis or Mycobacterium tuberculosisderived proteins. Certain adjuvants are commercially available as, forexample, Freund's Incomplete Adjuvant and Complete Adjuvant (DifcoLaboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company,Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.);aluminum salts such as aluminum hydroxide gel (alum) or aluminumphosphate; salts of calcium, iron or zinc; an insoluble suspension ofacylated tyrosine; acylated sugars; cationically or anionicallyderivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF, interleukin-2,-7,-12, and other like growth factors, may also beused as adjuvants.

[0139] Within certain embodiments of the invention, the adjuvantcomposition may be one that induces an immune response predominantly ofthe Th1 type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2and IL-12) tend to favor the induction of cell mediated immune responsesto an administered antigen. In contrast, high levels of Th2-typecytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the inductionof humoral immune responses. Following application of a vaccine asprovided herein, a patient will support an immune response that includesTh1- and Th2-type responses. Within an embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, Ann. Rev.Immunol. 7:145-173, 1989.

[0140] Certain adjuvants for eliciting a predominantly Th1-type responseinclude, for example, a combination of monophosphoryl lipid A, including3-de-O-acylated monophosphoryl lipid A, together with an aluminum salt.MPL® adjuvants are available from Corixa Corporation (Seattle, Wash;see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and4,912,094). CpG-containing oligonucleotides (in which the CpGdinucleotide is unmethylated) also induce a predominantly Th1 response.Such oligonucleotides are well known and are described, for example, inWO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462.Immunostimulatory DNA sequences are also described, for example, by Satoet al., Science 273:352, 1996. Another adjuvant comprises a saponin,such as Quil A, or derivatives thereof, including QS21 and QS7 (AquilaBiopharmaceuticals Inc., Framingham, Mass.); Escin; Digitonin; orGypsophila or Chenopodium quinoa saponins. Other formulations includemore than one saponin in the adjuvant combinations of the presentinvention, for example combinations of at least two of the followinggroup comprising QS21, QS7, Quil A, β-escin, or digitonin.

[0141] Alternatively the saponin formulations may be combined withvaccine vehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, liposomes and lipid-basedparticles, particles composed of glycerol monoesters, etc. The saponinsmay also be formulated in the presence of cholesterol to formparticulate structures such as liposomes or ISCOMs. Furthermore, thesaponins may be formulated together with a polyoxyethylene ether orester, in either a non-particulate solution or suspension, or in aparticulate structure such as a paucilamelar liposome or ISCOM. Thesaponins may also be formulated with excipients such as Carbopol^(R) toincrease viscosity, or may be formulated in a dry powder form with apowder excipient such as lactose.

[0142] In one embodiment, the adjuvant system includes the combinationof a monophosphoryl lipid A and a saponin derivative, such as thecombination of QS21 and 3D-MPL® adjuvant, as described in WO 94/00153,or a less reactogenic composition where the QS21 is quenched withcholesterol, as described in WO 96/33739. Other formulations comprise anoil-in-water emulsion and tocopherol. Another adjuvant formulationemploying QS21, 3D-MPL® adjuvant and tocopherol in an oil-in-wateremulsion is described in WO 95/17210.

[0143] Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative particularly thecombination of CpG and QS21 as disclosed in WO 00/09159. The formulationmay additionally comprise an oil in water emulsion and tocopherol.

[0144] Additional illustrative adjuvants for use in the pharmaceuticalcompositions of the invention include Montanide ISA 720 (Seppic,France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59(Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4,available from SmithKline Beecham, Rixensart, Belgium), Detox(Enhanzyn®) (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.)and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as thosedescribed in pending U.S. patent application Ser. Nos. 08/853,826 and09/074,720, the disclosures of which are incorporated herein byreference in their entireties, and polyoxyethylene ether adjuvants suchas those described in WO 99/52549A1.

[0145] Other adjuvants include adjuvant molecules of the general formula(I):

HO(CH₂CH₂O)_(n)-A-R

[0146] Wherein, n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl orPhenyl C₁₋₅₀ alkyl.

[0147] One embodiment of the present invention consists of a vaccineformulation comprising a polyoxyethylene ether of general formula (I),wherein n is between 1 and 50, or 4-24, or 9; the R component is C1-50,or C4-C20 alkyl or C12 alkyl, and A is a bond. The concentration of thepolyoxyethylene ethers should be in the range 0.1-20%, or from 0.1-10%,or in the range 0.1-1%. Polyoxyethylene ethers may be selected from thefollowing group: polyoxyethylene-9-lauryl ether,polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such aspolyoxyethylene lauryl ether are described in the Merck index (12thedition: entry 7717). These adjuvant molecules are described in WO99/52549.

[0148] The polyoxyethylene ether according to the general formula (I)above may, if desired, be combined with another adjuvant. For example,an adjuvant combination may be with CpG as described in the pending UKpatent application GB 9820956.2.

[0149] A carrier is also present in the vaccine composition according tothe invention. The carrier may be an oil in water emulsion, or analuminium salt, such as aluminium phosphate or aluminium hydroxide.

[0150] A oil-in-water emulsion may comprise a metabolisible oil, such assqualene, alpha tocopherol and Tween 80. In one aspect, the antigens inthe vaccine composition according to the invention are combined withQS21 and 3D-MPL in such an emulsion. Additionally the oil in wateremulsion may contain span 85 and/or lecithin and/or tricaprylin.

[0151] Typically for human administration QS21 and 3D-MPL will bepresent in a vaccine in the range of 1 μg-200 μg, such as 10-100 μg, or10 μg-50 μg per dose. Typically the oil in water will comprise from 2 to10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% tween80. The ratio of squalene may be: alpha tocopherol is equal to or lessthan 1 as this provides a more stable emulsion. Span 85 may also bepresent at a level of 1%. In some cases it may be advantageous that thevaccines of the present invention will further contain a stabiliser.

[0152] Non-toxic oil in water emulsions may contain a non-toxic oil,e.g. squalane or squalene, an emulsifier, e.g. Tween 80, in an aqueouscarrier. The aqueous carrier may be, for example, phosphate bufferedsaline.

[0153] A particularly potent adjuvant formulation involving QS21, 3D-MPLand tocopherol in an oil in water emulsion is described in WO 95/17210.

[0154] The present invention also provides a polyvalent vaccinecomposition comprising a vaccine formulation of the invention incombination with other antigens, in particular antigens useful fortreating cancers, more particularly colorectal cancer, autoimmunediseases and related conditions. Such a polyvalent vaccine compositionmay include a TH-1 inducing adjuvant as hereinbefore described.

[0155] According to another embodiment of this invention, an immunogeniccomposition described herein is delivered to a host via antigenpresenting cells (APCs), such as dendritic cells, macrophages, B cells,monocytes and other cells that may be engineered to be efficient APCs.Such cells may, but need not, be genetically modified to increase thecapacity for presenting the antigen, to improve activation and/ormaintenance of the T cell response, to have anti-tumor effects per seand/or to be immunologically compatible with the receiver (i.e., matchedHLA haplotype). APCs may generally be isolated from any of a variety ofbiological fluids and organs, including tumor and peritumoral tissues,and may be autologous, allogeneic, syngeneic or xenogeneic cells.

[0156] Certain embodiments of the present invention may use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529,1999). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in vitro), their ability to take up, process andpresent antigens with high efficiency and their ability to activatenaive T cell responses. Dendritic cells may, of course, be engineered toexpress specific cell-surface receptors or ligands that are not commonlyfound on dendritic cells in vivo or ex vivo, and such modified dendriticcells are contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within a vaccine (see Zitvogel et al.,Nature Med. 4:594-600, 1998).

[0157] Dendritic cells and progenitors may be obtained from peripheralblood, bone marrow, tumor-infiltrating cells, peritumoraltissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cordblood or any other suitable tissue or fluid. For example, dendriticcells may be differentiated ex vivo by adding a combination of cytokinessuch as GM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytesharvested from peripheral blood. Alternatively, CD34 positive cellsharvested from peripheral blood, umbilical cord blood or bone marrow maybe differentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

[0158] Dendritic cells are conveniently categorized as “immature” and“mature” cells, which allows a simple way to discriminate between twowell characterised phenotypes.

[0159] However, this nomenclature should not be construed to exclude allpossible intermediate stages of differentiation. Immature dendriticcells are characterised as APC with a high capacity for antigen uptakeand processing, which correlates with the high expression of Fcγreceptor and mannose receptor. The mature phenotype is typicallycharacterized by a lower expression of these markers, but a highexpression of cell surface molecules responsible for T cell activationsuch as class I and class II MHC, adhesion molecules (e.g., CD54 andCD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB).

[0160] APCs may generally be transfected with a polynucleotide of theinvention (or portion or other variant thereof) such that the encodedpolypeptide, or an immunogenic portion thereof, is expressed on the cellsurface. Such transfection may take place ex vivo, and a pharmaceuticalcomposition comprising such transfected cells may then be used fortherapeutic purposes, as described herein. Alternatively, a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to a patient, resulting in transfection thatoccurs in vivo. In vivo and ex vivo transfection of dendritic cells, forexample, may generally be performed using any methods known in the art,such as those described in WO 97/24447, or the gene gun approachdescribed by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.Antigen loading of dendritic cells may be achieved by incubatingdendritic cells or progenitor cells with the tumor polypeptide, DNA(naked or within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide.

[0161] While any suitable carrier known to those of ordinary skill inthe art may be employed in the pharmaceutical compositions of thisinvention, the type of carrier will typically vary depending on the modeof administration. Compositions of the present invention may beformulated for any appropriate manner of administration, including forexample, topical, oral, nasal, mucosal, intravenous, intracranial,intraperitoneal, subcutaneous and intramuscular administration.

[0162] Carriers for use within such pharmaceutical compositions arebiocompatible, and may also be biodegradable. In certain embodiments,the formulation may provide a relatively constant level of activecomponent release. In other embodiments, however, a more rapid rate ofrelease immediately upon administration may be desired. The formulationof such compositions is well within the level of ordinary skill in theart using known techniques. Illustrative carriers useful in this regardinclude microparticles of poly(lactide-co-glycolide), polyacrylate,latex, starch, cellulose, dextran and the like. Other illustrativedelayed-release carriers include supramolecular biovectors, whichcomprise a non-liquid hydrophilic core (e.g., a cross-linkedpolysaccharide or oligosaccharide) and, optionally, an external layercomprising an amphiphilic compound, such as a phospholipid (see e.g.,U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701and WO 96/06638). The amount of active compound contained within asustained release formulation depends upon the site of implantation, therate and expected duration of release and the nature of the condition tobe treated or prevented.

[0163] In another illustrative embodiment, biodegradable microspheres(e.g., polylactate polyglycolate) are employed as carriers for thecompositions of this invention. Suitable biodegradable microspheres aredisclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109;5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and5,942,252. Modified hepatitis B core protein carrier systems such asdescribed in WO/99 40934, and references cited therein, will also beuseful for many applications. Another illustrative carrier/deliverysystem employs a carrier comprising particulate-protein complexes, suchas those described in U.S. Pat. No. 5,928,647, which are capable ofinducing a class I-restricted cytotoxic T lymphocyte responses in ahost.

[0164] The pharmaceutical compositions of the invention will oftenfurther comprise one or more buffers (e.g., neutral buffered saline orphosphate buffered saline), carbohydrates (e.g., glucose, mannose,sucrose or dextrans), mannitol, proteins, polypeptides or amino acidssuch as glycine, antioxidants, bacteriostats, chelating agents such asEDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes thatrender the formulation isotonic, hypotonic or weakly hypertonic with theblood of a recipient, suspending agents, thickening agents and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilizate.

[0165] The pharmaceutical compositions described herein may be presentedin unit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are typically sealed in such a way to preserve thesterility and stability of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles. Alternatively, a pharmaceutical compositionmay be stored in a freeze-dried condition requiring only the addition ofa sterile liquid carrier immediately prior to use.

[0166] The development of suitable dosing and treatment regimens forusing the particular compositions described herein in a variety oftreatment regimens, including e.g., oral, parenteral, intravenous,intranasal, and intramuscular administration and formulation, is wellknown in the art, some of which are briefly discussed below for generalpurposes of illustration.

[0167] In certain applications, the pharmaceutical compositionsdisclosed herein may be delivered via oral administration to an animal.As such, these compositions may be formulated with an inert diluent orwith an assimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

[0168] The active compounds may even be incorporated with excipients andused in the form of ingestible tablets, buccal tables, troches,capsules, elixirs, suspensions, syrups, wafers, and the like (see, forexample, Mathiowitz et al., Nature Mar. 27, 1997; 386(6623):410-4; Hwanget al., Crit Rev Ther Drug Carrier Syst 1998;15(3):243-84; U.S. Pat. No.5,641,515; U.S. Pat. No. 5,580,579 and U.S. Pat. No. 5,792,451).Tablets, troches, pills, capsules and the like may also contain any of avariety of additional components, for example, a binder, such as gumtragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, alginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose or saccharinmay be added or a flavoring agent, such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar, or both.Of course, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparation and formulations.

[0169] Typically, these formulations will contain at least about 0.1% ofthe active compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

[0170] For oral administration the compositions of the present inventionmay alternatively be incorporated with one or more excipients in theform of a mouthwash, dentifrice, buccal tablet, oral spray, orsublingual orally-administered formulation. Alternatively, the activeingredient may be incorporated into an oral solution such as onecontaining sodium borate, glycerin and potassium bicarbonate, ordispersed in a dentifrice, or added in a therapeutically-effectiveamount to a composition that may include water, binders, abrasives,flavoring agents, foaming agents, and humectants. Alternatively thecompositions may be fashioned into a tablet or solution form that may beplaced under the tongue or otherwise dissolved in the mouth.

[0171] In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally. Suchapproaches are well known to the skilled artisan, some of which arefurther described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat.No. 5,641,515 and U.S. Pat. No. 5,399,363. In certain embodiments,solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations generally will contain a preservative to prevent the growthof microorganisms.

[0172] Illustrative pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (for example, see U.S. Pat. No. 5,466,468). In all cases theform must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and/or vegetable oils.Proper fluidity may be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants. The preventionof the action of microorganisms can be facilitated by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, isotonic agents may be included, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

[0173] In one embodiment, for parenteral administration in an aqueoussolution, the solution should be suitably buffered if necessary and theliquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. In this connection, a sterile aqueous medium that can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage may be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. Moreover, for humanadministration, preparations will meet sterility, pyrogenicity, and thegeneral safety and purity standards as required by FDA Office ofBiologics standards.

[0174] In another embodiment of the invention, the compositionsdisclosed herein may be formulated in a neutral or salt form.Illustrative pharmaceutically-acceptable salts include the acid additionsalts (formed with the free amino groups of the protein) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective.

[0175] The carriers can further comprise any and all solvents,dispersion media, vehicles, coatings, diluents, antibacterial andantifungal agents, isotonic and absorption delaying agents, buffers,carrier solutions, suspensions, colloids, and the like. The use of suchmedia and agents for pharmaceutical active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions. The phrase“pharmaceutically-acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human.

[0176] In certain embodiments, the pharmaceutical compositions may bedelivered by intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering genes, nucleic acids, andpeptide compositions directly to the lungs via nasal aerosol sprays hasbeen described, e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No.5,804,212. Likewise, the delivery of drugs using intranasalmicroparticle resins (Takenaga et al., J Controlled Release Mar. 2,1998;52(1-2):81-7) and lysophosphatidyl-glycerol compounds (U.S. Pat.No. 5,725,871) are also well-known in the pharmaceutical arts. Likewise,illustrative transmucosal drug delivery in the form of apolytetrafluoroetheylene support matrix is described in U.S. Pat. No.5,780,045.

[0177] In certain embodiments, liposomes, nanocapsules, microparticles,lipid particles, vesicles, and the like, are used for the introductionof the compositions of the present invention into suitable hostcells/organisms. In particular, the compositions of the presentinvention may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike. Alternatively, compositions of the present invention can be bound,either covalently or non-covalently, to the surface of such carriervehicles.

[0178] The formation and use of liposome and liposome-like preparationsas potential drug carriers is generally known to those of skill in theart (see for example, Lasic, Trends Biotechnol Jul. 16, 1998 (7):307-21;Takakura, Nippon Rinsho March 1998;56(3):691-5; Chandran et al., IndianJ Exp Biol. August 1997;35(8):801-9; Margalit, Crit Rev Ther DrugCarrier Syst. 1995;12(2-3):233-61; U.S. Pat. No. 5,567,434; U.S. Pat.No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S.Pat. No. 5,795,587, each specifically incorporated herein by referencein its entirety).

[0179] Liposomes have been used successfully with a number of cell typesthat are normally difficult to transfect by other procedures, includingT cell suspensions, primary hepatocyte cultures and PC 12 cells(Renneisen et al., J Biol Chem. Sep. 25, 1990; 265(27):16337-42; Mulleret al., DNA Cell Biol. Apr. 9, 1990; (3):221-9). In addition, liposomesare free of the DNA length constraints that are typical of viral-baseddelivery systems. Liposomes have been used effectively to introducegenes, various drugs, radiotherapeutic agents, enzymes, viruses,transcription factors, allosteric effectors and the like, into a varietyof cultured cell lines and animals. Furthermore, he use of liposomesdoes not appear to be associated with autoimmune responses orunacceptable toxicity after systemic delivery.

[0180] In certain embodiments, liposomes are formed from phospholipidsthat are dispersed in an aqueous medium and spontaneously formmultilamellar concentric bilayer vesicles (also termed multilamellarvesicles (MLVs).

[0181] Alternatively, in other embodiments, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (see, for example, Quintanar-Guerrero etal., Drug Dev Ind Pharm. Dec. 24, 1998; (12):1113-28). To avoid sideeffects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 μm) may be designed using polymers able tobe degraded in vivo. Such particles can be made as described, forexample, by Couvreur et al., Crit Rev Ther Drug Carrier Syst.1988;5(1):1-20; zur Muhlen et al., Eur J Pharm Biopharm. March 1998;45(2):149-55; Zambaux et al. J Controlled Release. Jan. 2,1998;50(1-3):31-40; and U.S. Pat. No. 5,145,684.

[0182] This invention also relates to the use of polynucleotides, in theform of primers derived from the polynucleotides of the presentinvention, and of polypeptides, in the form of antibodies or reagentsspecific for the polypeptide of the present invention, as diagnosticreagents.

[0183] The identification of genetic or biochemical markers in blood ortissues that will enable the detection of very early changes along thecarcinogenesis pathway will help in determining the best treatment forthe patient. Surrogate tumour markers, such as polynucleotideexpression, can be used to diagnose different forms and states ofcancer. The identification of expression levels of the polynucleotidesof the invention will be useful in both the staging of the cancerousdisorder and grading the nature of the cancerous tissue. The stagingprocess monitors the advancement of the cancer and is determined on thepresence or absence of malignant tissue in the areas biopsied. Thepolynucleotides of the invention can help to perfect the staging processby identifying markers for the aggresivity of a cancer, for example thepresence in different areas of the body. The grading of the cancerdescribes how closely a tumour resembles normal tissue of its same typeand is assessed by its cell morphology and other markers ofdifferentiation. The polynucleotides of the invention can be useful indetermining the tumour grade as they can help in the determination ofthe differentiation status of the cells of a tumour.

[0184] The diagnostic assays offer a process for diagnosing ordetermining a susceptibility to cancers, autoimmune disease and relatedconditions through diagnosis by methods comprising determining from asample derived from a subject an abnormally decreased or increased levelof polypeptide or mRNA. This method of diagnosis is known asdifferential expression. The expression of a particular gene is comparedbetween a diseased tissue and a normal tissue. A difference between thepolynucleotide-related gene, mRNA, or protein in the two tissues iscompared, for example in molecular weight, amino acid or nucleotidesequence, or relative abundance, indicates a change in the gene, or agene which regulates it, in the tissue of the human that was suspectedof being diseased.

[0185] Decreased or increased expression can be measured at the RNAlevel. PolyA RNA is first isolated from the two tissues and thedetection of mRNA encoded by a gene corresponding to a differentiallyexpressed polynucleotide of the invention can be detected by, forexample, in situ hybridization in tissue sections, reversetrascriptase-PCR, using Northern blots containing poly A+ mRNA, or anyother direct or inderect RNA detection method. An increased or decreasedexpression of a given RNA in a diseased tissue compared to a normaltissue suggests that the transcript and/or the expressed protein has arole in the disease. Thus detection of a higher or lower level of mRNAcorresponding to SEQ ID NO: 1 relative to normal level is indicative ofthe presence of cancer in the patient.

[0186] mRNA expression levels in a sample can be determined bygeneration of a library of expressed sequence tags (ESTs) from thesample. The relative representation of ESTs in the library can be usedto assess the relative representation of the gene transcript in thestarting sample. The EST analysis of the test can then be compared tothe EST analysis of a reference sample to determine the relativeexpression levels of the polynucleotide of interest.

[0187] Other mRNA analyses can be carried out using serial analysis ofgene expression (SAGE) methodology (Velculescu et. Al. Science (1995)270:484), differential display methodology (For example, U.S. Pat. No.5,776,683) or hybridization analysis which relies on the specificity ofnucleotide interactions.

[0188] Alternatively, the comparison could be made at the protein level.The protein sizes in the two tissues may be compared using antibodies todetect polypeptides in Western blots of protein extracts from the twotissues. Expression levels and subcellular localization may also bedetected immunologically using antibodies to the corresponding protein.Further assay techniques that can be used to determine levels of aprotein, such as a polypeptide of the present invention, in a samplederived from a host are well-known to those of skill in the art. Araised or decreased level of polypeptide expression in the diseasedtissue compared with the same protein expression level in the normaltissue indicates that the expressed protein may be involved in thedisease.

[0189] In the assays of the present invention, the diagnosis can bedetermined by detection of gene product expression levels encoded by atleast one sequence set forth in SEQ ID NO: 1. A comparison of the mRNAor protein levels in a diseased versus normal tissue may also be used tofollow the progression or remission of a disease.

[0190] A large number of polynucleotide sequences in a sample can beassayed using polynucleotide arrays. These can be used to examinedifferential expression of genes and to determine gene function. Forexample, arrays of the polynucleotide sequences SEQ ID NO:1 can be usedto determine if any of the polynucleotides are differentially expressedbetween a normal and cancer cell. In one embodiment of the invention, anarray of oligonucleotides probes comprising the SEQ ID NO:1 nucleotidesequence or fragments thereof can be constructed to conduct efficientscreening of e.g., genetic mutations. Array technology methods are wellknown and have general applicability and can be used to address avariety of questions in molecular genetics including gene expression,genetic linkage, and genetic variability (see for example: M. Chee etal., Science, Vol 274, pp 610-613 (1996)).

[0191] “Diagnosis” as used herein includes determination of a subject'ssusceptibility to a disease, determination as to whether a subjectpresently has the disease, and also the prognosis of a subject affectedby the disease.

[0192] The present invention, further relates to a diagnostic kit forperforming a diagnostic assay which comprises:

[0193] (a) a polynucleotide of the present invention, which may be thenucleotide sequence of SEQ ID NO: 1, or a fragment thereof;

[0194] (b) a nucleotide sequence complementary to that of (a), which maybe the nucleotide sequence of SEQ ID NO: 6;

[0195] (c) a polypeptide of the present invention, which may be thepolypeptide of SEQ ID NO: 2 or 3, or a fragment thereof; or

[0196] (d) an antibody to a polypeptide of the present invention, whichmay be the polypeptide of SEQ ID NO:2 or 3.

[0197] The nucleotide sequences of the present invention are alsovaluable for chromosomal localisation. The sequence is specificallytargeted to, and can hybridize with, a particular location on anindividual human chromosome. The mapping of relevant sequences tochromosomes according to the present invention is an important firststep in correlating those sequences with gene associated disease. Once asequence has been mapped to a precise chromosomal location, the physicalposition of the sequence on the chromosome can be correlated withgenetic map data. Such data are found in, for example, V. McKusick,Mendelian Inheritance in Man (available on-line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).The differences in the cDNA or genomic sequence betweenaffected and unaffected individuals can also be determined.

[0198] The polypeptides of the invention or their fragments or analogsthereof, or cells expressing them, can also be used as immunogens toproduce antibodies immunospecific for polypeptides of the presentinvention. The term “immunospecific” means that the antibodies havesubstantially greater affinity for the polypeptides of the inventionthan their affinity for other related polypeptides in the prior art.

[0199] In a further aspect the invention provides an antibodyimmunospecific for a polypeptide according to the invention or animmunological fragment thereof as hereinbefore defined. The antibody maybe a monoclonal antibody.

[0200] Antibodies generated against polypeptides of the presentinvention may be obtained by administering the polypeptides orepitope-bearing fragments, analogs or cells to an animal, which may be anon-human animal, using routine protocols. For preparation of monoclonalantibodies, any technique which provides antibodies produced bycontinuous cell line cultures can be used. Examples include thehybridoma technique (Kohler, G. and Milstein, C., Nature (1975)256:495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., Immunology Today (1983) 4:72) and the EBV-hybridomatechnique (Cole et al., Monoclonal Antibodies and Cancer Therapy, 77-96,Alan R. Liss, Inc., 1985).

[0201] Techniques for the production of single chain antibodies, such asthose described in U.S. Pat. No. 4,946,778, can also be adapted toproduce single chain antibodies to polypeptides of this invention. Also,transgenic mice, or other organisms, including other mammals, may beused to express humanized antibodies.

[0202] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptide or to purify the polypeptidesby affinity chromatography. The antibody of the invention may also beemployed to prevent or treat cancer, particularly colorectal cancer,autoimmune disease and related conditions.

[0203] Another aspect of the invention relates to a method for inducingor modulating an immunological response in a mammal which comprisesinoculating the mammal with a polypeptide of the present invention,adequate to produce antibody and/or T cell immune response to protect orameliorate the symptoms or progression of the disease. Yet anotheraspect of the invention relates to a method of inducing or modulatingimmunological response in a mammal which comprises, delivering apolypeptide of the present invention via a vector directing expressionof the polynucleotide and coding for the polypeptide in vivo in order toinduce such an immunological response to produce antibody to protectsaid animal from diseases.

[0204] It will be appreciated that the present invention thereforeprovides a method of treating abnormal conditions such as, for instance,cancer and autoimmune diseases, in particular, colorectal cancer,related to either a presence of, an excess of, or an under-expressionof, CASB7439 polypeptide activity. Other abnormal conditions related toCASB7439 expression that the invention seeks to treat are chroniclymphocytic leukemiae and germ cell tumours.

[0205] The present invention further provides for a method of screeningcompounds to identify those which stimulate or which inhibit thefunction of the CASB7439 polypeptide. In general, agonists orantagonists may be employed for therapeutic and prophylactic purposesfor such diseases as hereinbefore mentioned. Compounds may be identifiedfrom a variety of sources, for example, cells, cell-free preparations,chemical libraries, and natural product mixtures. Such agonists,antagonists or inhibitors so-identified may be natural or modifiedsubstrates, ligands, receptors, enzymes, etc., as the case may be, ofthe polypeptide; or may be structural or functional mimetics thereof(see Coligan et al., Current Protocols in Immunology 1(2):Chapter 5(1991)). Screening methods will be known to those skilled in the art.Further screening methods may be found in for example D. Bennett et al.,J Mol Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459-9471 (1995) and references therein.

[0206] Thus the invention provides a method for screening to identifycompounds which stimulate or which inhibit the function of thepolypeptide of the invention which comprises a method selected from thegroup consisting of:

[0207] (a) measuring the binding of a candidate compound to thepolypeptide (or to the cells or membranes bearing the polypeptide) or afusion protein thereof by means of a label directly or indirectlyassociated with the candidate compound;

[0208] (b) measuring the binding of a candidate compound to thepolypeptide (or to the cells or membranes bearing the polypeptide) or afusion protein thereof in the presense of a labeled competitior;

[0209] (c) testing whether the candidate compound results in a signalgenerated by activation or inhibition of the polypeptide, usingdetection systems appropriate to the cells or cell membranes bearing thepolypeptide;

[0210] (d) mixing a candidate compound with a solution containing apolypeptide of claim 1, to form a mixture, measuring activity of thepolypeptide in the mixture, and comparing the activity of the mixture toa standard; or

[0211] (e) detecting the effect of a candidate compound on theproduction of mRNA encoding said polypeptide and said polypeptide incells, using for instance, an ELISA assay.

[0212] The polypeptide of the invention may be used to identify membranebound or soluble receptors, if any, through standard receptor bindingtechniques known in the art.

[0213] Well known screening methods may also be used to identifyagonists and antagonists of the polypeptide of the invention whichcompete with the binding of the polypeptide of the invention to itsreceptors, if any.

[0214] Thus, in another aspect, the present invention relates to ascreening kit for identifying agonists, antagonists, ligands, receptors,substrates, enzymes, etc. for polypeptides of the present invention; orcompounds which decrease or enhance the production of such polypeptides,which comprises:

[0215] (a) a polypeptide of the present invention;

[0216] (b) a recombinant cell expressing a polypeptide of the presentinvention;

[0217] (c) a cell membrane expressing a polypeptide of the presentinvention; or

[0218] (d) antibody to a polypeptide of the present invention;

[0219] which polypeptide may be that of SEQ ID NO:2 or 3.

[0220] It will be readily appreciated by the skilled artisan that apolypeptide of the present invention may also be used in a method forthe structure-based design of an agonist, antagonist or inhibitor of thepolypeptide, by:

[0221] (a) determining in the first instance the three-dimensionalstructure of the polypeptide;

[0222] (b) deducing the three-dimensional structure for the likelyreactive or binding site(s) of an agonist, antagonist or inhibitor;

[0223] (c) synthesing candidate compounds that are predicted to bind toor react with the deduced binding or reactive site; and

[0224] (d) testing whether the candidate compounds are indeed agonists,antagonists or inhibitors.

[0225] Gene therapy may also be employed to effect the endogenousproduction of CASB7439 polypeptide by the relevant cells in the subject.For an overview of gene therapy, see Chapter 20, Gene Therapy and otherMolecular Genetic-based Therapeutic Approaches, (and references citedtherein) in Human Molecular Genetics, T Strachan and A P Read, BIOSScientific Publishers Ltd (1996).

[0226] Vaccine preparation is generally described in PharmaceuticalBiotechnology, Vol.61 Vaccine Design—the subunit and adjuvant approach,edited by Powell and Newman, Plenum Press, 1995. New Trends andDevelopments in Vaccines, edited by Voller et al., University ParkPress, Baltimore, Md., U.S.A. 1978. Encapsulation within liposomes isdescribed, for example, by Fullerton, U.S. Pat. No. 4,235,877.Conjugation of proteins to macromolecules is disclosed, for example, byLikhite, U.S. Pat. No. 4,372,945 and by Armor et al., U.S. Pat. No.4,474,757.

[0227] The amount of protein in each vaccine dose is selected as anamount which induces an immunoprotective response without significant,adverse side effects in typical vaccinees. Such amount will varydepending upon which specific immunogen is employed. Generally, it isexpected that each dose will comprise 1-1000 μg of protein, or 2-100 μg,or 4-40 μg. An optimal amount for a particular vaccine can beascertained by standard studies involving observation of antibody titresand other responses in subjects. Following an initial vaccination,subjects may receive a boost in about 4 weeks.

[0228] “Isolated” means altered “by the hand of man” from the naturalstate. If an “isolated” composition or substance occurs in nature, ithas been changed or removed from its original environment, or both. Forexample, a polynucleotide or a polypeptide naturally present in a livinganimal is not “isolated,” but the same polynucleotide or polypeptideseparated from the coexisting materials of its natural state is“isolated”, as the term is employed herein.

[0229] “Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA including single and double stranded regions.

[0230] “Variant” refers to a polynucleotide or polypeptide that differsfrom a reference polynucleotide or polypeptide, but retains essentialproperties. A typical variant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions in any combination. A substituted orinserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

[0231] “Identity,” as known in the art, is a relationship between two ormore polypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Methods to determine identity may be designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Computer program methods to determine identity and similaritybetween two sequences include, but are not limited to, the GCG programpackage (Devereux, J., et al., Nucleic Acids Research 12(1): 387(1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well known Smith Waterman algorithm may also be usedto determine identity.

[0232] The algorithm used may be FASTA. The parameters for polypeptideor polynuleotide sequence comparison using this algorithm include thefollowing:

[0233] Gap Penalty: 12

[0234] Gap extension penalty: 4

[0235] Word size: 2, max 6

[0236] Parameters for polypeptide sequence comparison with other methodsinclude the following:

[0237] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453(1970)

[0238] Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci. USA. 89:10915-10919 (1992)

[0239] Gap Penalty: 12

[0240] Gap Length Penalty: 4

[0241] A program useful with these parameters is publicly available asthe “gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for polypeptidecomparisons (along with no penalty for end gaps).

[0242] Parameters for polynucleotide comparison include the following:

[0243] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453(1970)

[0244] Comparison matrix: matches=+10, mismatch=0

[0245] Gap Penalty: 50

[0246] Gap Length Penalty: 3

[0247] A program useful with these parameters is publicly available asthe “gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for polynucleotidecomparisons.

[0248] By way of example, a polynucleotide sequence of the presentinvention may be identical to the reference sequence of SEQ ID NO:1,that is be 100% identical, or it may include up to a certain integernumber of nucleotide alterations as compared to the reference sequence.Such alterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofnucleotide alterations is determined by multiplying the total number ofnucleotides in SEQ ID NO:1 by the numerical percent of the respectivepercent identity(divided by 100) and subtracting that product from saidtotal number of nucleotides in SEQ ID NO:1, or:

n _(n) ≦x _(n)−(x _(n) ·y)

[0249] wherein n_(n) is the number of nucleotide alterations, x_(n) isthe total number of nucleotides in SEQ ID NO:1, and y is, for instance,0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%,etc., and wherein any non-integer product of x_(n) and y is rounded downto the nearest integer prior to subtracting it from x_(n). Alterationsof a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 maycreate nonsense, missense or frameshift mutations in this codingsequence and thereby alter the polypeptide encoded by the polynucleotidefollowing such alterations.

[0250] Similarly, a polypeptide sequence of the present invention may beidentical to the reference sequence of SEQ ID NO:2, that is be 100%identical, or it may include up to a certain integer number of aminoacid alterations as compared to the reference sequence such that the %identity is less than 100%. Such alterations are selected from the groupconsisting of at least one amino acid deletion, substitution, includingconservative and non-conservative substitution, or insertion, andwherein said alterations may occur at the amino- or carboxy-terminalpositions of the reference polypeptide sequence or anywhere betweenthose terminal positions, interspersed either individually among theamino acids in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of amino acidalterations for a given % identity is determined by multiplying thetotal number of amino acids in SEQ ID NO:2 by the numerical percent ofthe respective percent identity(divided by 100) and then subtractingthat product from said total number of amino acids in SEQ ID NO:2, or:

n _(a) ≦x _(a)−(x _(a) ·y),

[0251] wherein n_(a) is the number of amino acid alterations, x_(a) isthe total number of amino acids in SEQ ID NO:2, and y is, for instance0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein anynon-integer product of x_(a) and y is rounded down to the nearestinteger prior to subtracting it from x_(a).

[0252] “Homolog” is a generic term used in the art to indicate apolynucleotide or polypeptide sequence possessing a high degree ofsequence relatedness to a subject sequence. Such relatedness may bequntified by determining the degree of identity and/or similaritybetween the sequences being compared as hereinbefore described. Fallingwithin this generic term are the terms “ortholog”, meaning apolynucleotide or polypeptide that is the functional equivalent of apolynucleotide or polypeptide in another species and “paralog” meaning afunctionally similar sequence when considered within the same species.

[0253] “CASB7439 polypeptide” as used herein includes but is not limitedto isolated polypeptides comprising an amino acid sequence which has atleast 70% identity, or at least 80% identity, or at least 90% identity,or at least 95% identity, or at least 97-99% identity, to that of SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12or SEQ ID NO:14 each over the entire length of the respective sequence.Such polypeptides include, but are not limited to, those comprising theamino acid of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10 and SEQ ID NO:11.

[0254] “Immunogenicity” as used herein means an ability of a compound toinduce, enhance or increase antibody production, or induce, enhance orincrease a cellular immune response in an animal or immune cell, whereinsaid compound may be, but is not limited to, a polynucleotide,polypeptide, polypeptides fragment, and varients thereof.

[0255] “Immunoresponse” as used herein includes, but is not limited to,a human or animal's ability to produce antibodies or exhibit a immunecell response to an antigen.

[0256] The following examples illustrate various aspects of thisinvention. These examples do not limit the scope of this invention whichis defined by the appended claims.

EXAMPLES Example 1 Real-Time RT-PCR Analysis in Matched Tumour andNormal Colon Tissues from Multiple Patients

[0257] Real-time RT-PCR (U. Gibson. 1996. Genome Research: 6,996) isused to compare mRNA transcript abundance of the candidate antigen inmatched tumour and normal colon tissues from multiple patients. Inaddition, mRNA levels of the candidate gene in a panel of normal tissuesare also evaluated by this approach.

[0258] Total RNA from normal and tumour colon is extracted from snapfrozen biopsies using TriPure reagent (Boehringer). Total RNA fromnormal tissues is purchased from InVitrogen or is extracted from snapfrozen biopsies using TriPure reagent. Poly-A+ mRNA is purified fromtotal RNA after DNAase treatment using oligo-dT magnetic beads (Dynal).Quantification of the mRNA is performed by spectrofluorimetry(VersaFluor, BioRad) using SybrII dye (Molecular Probes). Primers forreal-time PCR amplification are designed with the Perkin-Elmer PrimerExpress software using default options for TaqMan amplificationconditions.

[0259] Real-time reactions are assembled according to standard PCRprotocols using 2 ng of purified mRNA for each reaction. SybrI dye(Molecular Probes) is added at a final dilution of {fraction (1/75,000)}for real-time detection. Amplification (40 cycles) and real-timedetection is performed in a Perkin-Elmer Biosystems PE7700 system usingconventional instrument settings. Ct values are calculated using thePE7700 Sequence Detector software. Several Ct values are obtained foreach samples: for the patient samples, the tumour Ct (CtT) and thematched normal colon Ct (CtN) values on the candidate TAA, and for thepanel of normal tissue samples, a CtXY for each normal tissue XY. Ananother Ct (CtA) is also calculated on Actin gene, as an internalreference, for all of the samples. Alternatively, real-time PCRamplification can be monitored using a Taqman probe. Amplification (40cycles) and real-time detection is performed in a Perkin-ElmerBiosystems PE7700 system using conventional instrument settings. Ctvalues are calculated using the PE7700 Sequence Detector Software. Ctvalues are obtained from each tissue sample for the target mRNA (CtX)and for the actin mRNA (CtA).

[0260] As the efficiency of PCR amplification under the prevailingexperimental conditions is close to the theoretical amplificationefficiency, 2^((CtN/T/XY-CtA)) value is an estimate of the relative TAAtranscript level of the sample, standardised with respect to Actintranscript level. A value of 1 thus suggests the candidate antigen andActin have the same expression level.

[0261] Real-time PCR reactions were first performed on tumour colon andmatching normal colon from biopsies of 12 patients (see Table 1).Reactions were then performed on a more complete data set totalling 18patients (see Table 2) (are included in this data set the first 12patients). Duplicates for 6 out of these 18 patients were made in thisdata set. Six further patients were tested, and the results were pooledwith the previous 18. The statistics on the final pool are shown inTable 3, and illustrated in FIG. 1.

[0262] A series of 48 normal tissue samples, representing 29 differenttissues, were also tested by the same procedure (analysed normal tissuesare given in able 3). TAA transcript levels are calculated as describedabove. The proportion of patients over-expressing the candidate antigen,as well as the average transcript over-expression versus normal tissuesis also calculated from this data set. The results are illustrated inFIG. 1. TABLE 1 CASB7439 Real-time PCR expression results: data set of12 patients. % of patients with a mRNA level higher in matched 92% tumorcolon (positive patients) % of patients with a mRNA level at least 3fold 92% higher in matched tumor colon % of patients with a mRNA levelat least 10 fold 92% higher in matched tumor colon % of patients with amRNA level at least 3 fold  8% lower in matched tumor colon. Averagematched normal colon mRNA 0.0026 level (Actin standardised) Averagematched tumor colon mRNA level 0.265 in positive patients (Actinstandardised) Average mRNA over-expression fold 2028 Median mRNAover-expression fold 115 Average normal tissues mRNA level 0.0079 Mediannormal tissues mRNA level 0.0016 Average normal tissues mRNA level0.0064 Median normal tissues mRNA level 0.0017 % of patients with a mRNAlevel higher than 92% average normal tissues % of patients with a mRNAlevel higher than 75% 10 fold average normal tissues Normalnon-dispensable tissues higher than None median normal tissue mRNA level

[0263] TABLE 2 CASB7439 Real-time PCR expression results: data set of 18patients. % of patients with a mRNA level higher in 89% matched tumorcolon (positive patients) % of patients with a mRNA level at least 3fold 89% higher in matched tumor colon % of patients with a mRNA levelat least 10 fold 78% higher in matched tumor colon % of patients with amRNA level at least 3 fold  5% lower in matched tumor colon. Averagematched normal colon mRNA level 0.005 (Actin standardised) Averagematched tumor colon mRNA level in 0.152 positive patients (Actinstandardised) Average mRNA over-expression fold 1100 Median mRNAover-expression fold 60 Average normal tissues mRNA level 0.0065 Mediannormal tissues mRNA level 0.0015 Average normal tissues mRNA level 0.005Median normal tissues mRNA level 0.0015 % of patients with a mRNA levelhigher than 94% median normal tissue % of patients with a mRNA levelhigher than 94% 10 fold median normal tissues Normal non-dispensabletissues higher than None median normal tissue mRNA level

[0264] TABLE 3 CASB7439 Real-time PCR expression results: data set of 24patients % of patients with a CASB7439 transcript level higher   92% intumor colon than adjacent normal colon (positive patients) % of positivepatients with a CASB7439 transcript level at least   75% 10 fold higherin tumor colon than adjacent normal colon Average transcriptover-expression fold in tumors 1289 of positive patients % of patientswith a CASB7439 transcript level higher in   96% tumor colon than normaltissue average % of patients with a mRNA level at least 10 fold higher62.5% in tumor colon than normal tissue average Normal tissues whereCASB7439 transcript expression none is equivalent to tumor transcriptlevel in tumors

[0265] Real-time PCR reactions were also performed using the Taqmanprotocol (as described above) on tumour colon and adjacent normal colonfrom biopsies of 6 patients (see Table 4). Three replicate measures weretaken for each, and the average was used for further calculations.Results are shown in FIG. 1. Moreover, 36 normal tissue samples,representing 28 different tissues (see Table 5), were also tested by thesame procedure. Results are shown in FIG. 2. TABLE 4 CASB7439 Real-timePCR expression results using Taqman probe Number of tumor samples fromdifferent patients 6 % of patients with a CASB7439 transcript level 100%higher in tumor colon than adjacent normal colon (positive patients) %of positive patients with a CASB7439 transcript level at least  83% 10fold higher in tumor colon than adjacent normal colon Average transcriptover-expression fold in tumors of 109 positive patients % of patientswith a CASB7439 transcript level higher in 100% tumor colon than normaltissue average % of patients with a mRNA level at least 10 fold higherin 100% tumor colon than normal tissue average Normal tissues whereCASB7439 transcript expression none is equivalent to tumor transcriptlevel in tumors

[0266] The results clearly suggest CASB7439 transcript is over-expressedin colorectal tumours compared to adjacent normal colon and to all ofthe above mentioned normal tissues. More than 90% of the patientsstrongly over-express CASB7439 transcript in tumour, as compared toadjacent normal colon. Average over-expression fold in the tumors is atleast of 100. Moreover, more than 90% of the patients over-express theCASB7439 transcript in colorectal tumors as compared to other normaltissues, more than 60% of them over-expressing it at least 10 fold.TABLE 5 listing of normal tissues used for CASB7439 transcriptexpression analysis. Tissue Abbreviation Adrenal gland Ad_Gl Aorta AoBladder Bl Bone marrow Bo_Ma Brain Bra Cervix Ce Colon Co Fallopian tubeFa_Tu Heart He Ileon Il Kidney Ki Liver Li Lung Lu Lymph node Ly_NoOesophagus Oe Parathyroid gland Pa_Thy Rectum Re Skin Sk Skeletal muscleSk_Mu Small intestine Sm_In Spleen Sp Stomach St Thyroid gland ThyTrachea Tra Ovary Ov Placenta Pl Prostate Pr Testis Te

Example 2 Differential Screening of cDNA Arrays

[0267] Identification of tumour-associated genes in the subtracted cDNAlibrary is accomplished by differential screening.

[0268] Total bacterial DNA is extracted from 100 μl over-night cultures.Bacteria are lysed with guanidium isothiocyantate and the bacterial DNAis affinity purified using magnetic glass (Boehringer). Plasmid insertsare recovered from the bacterial DNA by Advantage PCR amplification(Clontech). The PCR products are dotted onto two nylon membranes toproduce high density cDNA arrays using the Biomek 96 HDRT tool(Beekman). The spotted cDNA is covalently linked to the membrane by UVirradiation. The first membrane is hybridised with a mixed cDNA probeprepared from the tumour of a single patient. The second membrane ishybridised with an equivalent amount of mixed cDNA probe prepared fromnormal colon of the same patient. The probe cDNA is prepared by PCRamplification as described above and is labelled using the AlkPhosDirect System (Amersham). Hybridisation conditions and stringency washesare as described in the AlkPhos Direct kit. Hybridized probe is detectedby chemiluminescence. Hybridisation intensities for each cDNA fragmenton both blots are measured by film densitometry or direct measurement(BioRad Fluor-S Max). The ratio of the tumour to normal hybridisationintensities (T/N) is calculated for each gene to evaluate the degree ofover-expression in the tumour. Genes which are significantlyover-expressed in colon tumours are followed-up. Significance isarbitrarily defined as one standard deviation of the T/N frequencydistribution. Differential screening experiments are repeated using RNAfrom multiple patient donors (>18) to estimate the frequency ofover-expressing tumours in the patient population.

[0269] In addition, the DNA arrays are hybridised with mixed cDNA probesfrom normal tissues other than colon (see list above) to determine thelevel of expression of the candidate gene in these tissues.

Example 3 DNA Microarrays

[0270] DNA micro-arrays are used to examine mRNA expression profiles oflarge collections of genes in multiple samples. This information is usedto complement the data obtained by real-time PCR and provides anindependent measure of gene expression levels in tumors and normaltissues.

[0271] Examples of current technologies for production of DNAmicro-arrays include 1) The Affymetrix “GeneChip” arrays in whicholigonucleotides are synthetized on the surface of the chip by solidphase chemical synthesis using a photolithographic process 2) DNAspotting technology in which small volumes of a DNA solution arerobotically deposited and then immobilized onto the surface of a solidphase (e.g. glass). In both instances, the chips are hybridized withcDNA or cRNA which has been extracted from the tissue of interest (e.g.normal tissue, tumour etc . . . ) and labeled with radioactivity or witha fluorescent reporter molecule. The labeled material is hybridized tothe chip and the amount of probe bound to each sequence on the chip isdetermined using a specialized scanner. The experiment can be set-upwith a single fluorescent reporter (or radioactivity) or, alternatively,can be performed using two fluorescent reporters. In this latter case,each of the two samples is labeled with one of the reporter molecules.The two labeled samples are then hybridized competitively to thesequences on the DNA chip. The ratio of the two fluorescent signals isdetermined for each sequence on the chip. This ratio is used tocalculate the relative abundance of the transcript in the two samples.Detailed protocols are available from a number of sources including “DNAMicroarrays: A practical approach. Schena M. Oxford University Press1999” and the World Wide Web(http://cmgm.stanford.edu/pbrown/protocols/index.html),http://arrayit.com/DNA-Microarray-Protocols/) and specializeddistributors (e.g. Affymetrix).

Example 5 Northern-Southern Blot Analysis

[0272] Limited amounts of mixed tumour and matched normal colon cDNA areamplified by Advantage PCR (see above). Messenger RNA from multiplenormal tissues is also amplified using the same procedure. The amplifiedcDNA (1 μg) is electrophoresed on a 1.2% agarose gel and transferredonto a nylon membrane. The membrane is hybridised (AlkPhos DirectSystem) with a probe prepared using a fragment of the candidate TAAcDNA. Northern-Southern analysis provides information on transcriptsize, presence of splice variants and transcript abundance in tumour andnormal tissues.

Example 6 Northern Blot Analysis

[0273] Northern blots are produced according to standard protocols using1 μg of poly A+ mRNA. Radioactive probes are prepared using theReady-to-Go system (Pharmacia).

Example 7 Experimental Identification of the Full Length cDNA Sequence

[0274] Colon tumour cDNA libraries are constructed using the Lambda ZapII system (Stratagene) from 5 μg of polyA+ mRNA. The supplied protocolis followed except that SuperscriptII (Life Technologies) is used forthe reverse transcription step. Oligo dT-primed and random-primedlibraries are constructed. About 1.5×10⁶ independent phages are platedfor each screening of the library. Phage plaques are transferred ontonylon filters and hybridised using a cDNA probe labelled with AlkPhosDirect. Positive phages are detected by chemiluminescence. Positivephage are excised from the agar plat, eluted in 500 μl SM buffer andconfirmed by gene-specific PCR. Eluted phages are converted to singlestrand M13 bacteriophage by in vivo excision. The bacteriophage is thenconverted to double strand plasmid DNA by infection of E. coli. Infectedbacteria are plated and submitted to a second round of screening withthe cDNA probe. Plasmid DNA is purified from positive bacterial clonesand sequenced on both strands.

[0275] When the full length gene cannot be obtained directly from thecDNA library, missing sequence is isolated using RACE technology(Marathon Kit, ClonTech.). This approach relies on reverse transcribingmRNA into double strand cDNA, ligating linkers onto the ends of the cDNAand amplifying the desired extremity of the cDNA using a gene-specificprimer and one of the linker oligonucleotides. Marathon PCR products arecloned into a plasmid (pCRII-TOPO, InVitrogen) and sequenced.

[0276] The polynucleotide of SEQ ID NO:1 was obtained using thisprocedure.

Example 8 EST Profiles

[0277] A complementary approach to experimental antigen tissueexpression characterization is to explore the human EST database. ESTs(‘Expressed Sequence Tags) are small fragments of cDNA made from acollection of mRNA extracted from a particular tissue or cell line. Suchdatabase currently provides a massive amount of human ESTs (2 10⁶) fromseveral thousands of cDNA tissue libraries, including tumoral tissuesfrom various types and states of disease. By means of informatics tools(Blast), a comparison search of the CASB7439 sequence is performed inorder to have further insight into tissue expression. EST GenBankAccession number EST cDNA tissue library C00634 Human adult (K. Okubo)AA468668 NCI_CGAP_Co3 AA565752 NCI_CGAP_Co11 AA565766 NCI_CGAP_Co11AA565767 NCI_CGAP_Co11 AI337239 NCI_CGAP_Co16 AI337448 NCI_CGAP_Co16AI393930 NCI_CGAP_CLL1 AI473673 NCI_CGAP_Co14 AI632444 NCI_CGAP_GC6AI861937 NCI_CGAP_Co16 AI825214 NCI_CGAP_GC6 AW080652 NCI_CGAP_Co19AW083899 NCI_CGAP_Co19 AW206058 NCI_CGAP_Sub3 AW237006 NCI_CGAP_GC6AW364626 DT0036 AW449612 NCI_CGAP_Sub5

[0278] These ESTs match perfectly with CASB7439. The list contains 9ESTs from 4 different tumor colon libraries, one EST from one normalcolon library, 3 ESTs from one tumor germ cell library, one EST from onechronic lymphocyte leukemia cells library, 2 ESTs from 2 mixed tumorslibraries, 2 ESTs from libraries of unknown type. These results clearlysuggests, as expected, that CASB7439 is over-expressed in tumor tissues,as compared to normal tissues.

Example 9 9.1 Expression and Purification of Tumour-Specific Antigens

[0279] Expression in microbial hosts, or alternatively in vitrotranscription/translation, is used to produce the antigen of theinvention for vaccine purposes and to produce protein fragments or wholeprotein for rapid purification and generation of antibodies needed forcharacterization of the naturally expressed protein byimmunohistochemistry or for follow-up of purification.

[0280] Recombinant proteins may be expressed in two microbial hosts, E.coli and in yeast (such as Saccharomyces cerevisiae or Pichia pastoris).This allows the selection of the expression system with the bestfeatures for this particular antigen production. In general, therecombinant antigen will be expressed in E. coli and the reagent proteinexpressed in yeast.

[0281] The expression strategy first involves the design of the primarystructure of the recombinant antigen. In general an expression fusionpartner (EFP) is placed at the N terminal extremity to improve levels ofexpression that could also include a region useful for modulating theimmunogenic properties of the antigen, an immune fusion partner (IFP).In addition, an affinity fusion partner (AFP) useful for facilitatingfurther purification is included at the C-terminal end.

[0282] As mentioned above, several constructs might undergo comparativeevaluation: For rapid expression and purification as well as generationof antibodies against CASB7439, it is proposed to generate in E. coli afull length CASB7439 protein with NS1 as EFP and a histidine tail asAFP.

[0283] Therefore, two constructs are proposed:

[0284] Construct 1: Full length wild type CASB7439 cDNA in fusion withNS1 cDNA as EFP and with a histidine tail coding cDNA as an AFP (SEQ IDNO:8). The encoded fusion protein sequence is SEQ ID NO:10.

[0285] Construct 2: Full length mutated CASB7439 cDNA in fusion with NS1cDNA as EFP and with a histidine tail coding cDNA as an AFP (SEQ IDNO:9). It is proposed in this construct to have the first 50 codons ofnative CASB7439 cDNA replaced by codons specific of the E. coli codonusage, to enhance expression potential of CASB7439 in its E. coli host.The encoded fusion protein sequence is SEQ ID NO:10.

[0286] The CASB7439 protein design is as shown below:

[0287] “NS1” is the N-terminal fragment (80 mino acids) of the Influenzaprotein NS1. “HIS” is a polyhistidine tail.

[0288] The recombinant strain used is AR58: a cryptic λ lysogen derivedfrom N99 that is gal E::Tn 10,Δ-8(chlD-pgl),Δ-H1(cro-chlA),N+, and cI857(Proc. Natl. Acad. Sci. USA vol82, pp.88-92, January 1985 Biochemistry)

[0289] When the recombinant strains are available, the recombinantproduct is characterized by the evaluation of the level of expressionand the prediction of further solubility of the protein by analysis ofthe behavior in the crude extract.

[0290] After growth on appropriate culture medium and induction of therecombinant protein expression, total extracts are analyzed by SDS-PAGE.The recombinant proteins are visualized in stained gels and identifiedby Western blot analysis using specific antibodies.

[0291] Plasmid: name: TCM 281 pRIT . . . 15143 replicon: pMB1 selection:Kan promotor: PL long insert: NS1-C74-39-His

[0292] Expression of the Recombinant Protein from Construct 1:

[0293] Bacteria was grown in LB medium +50 μg/ml Kan at 30° C.

[0294] When the culture reached OD=0.5 (620 nm), the culture was heatedup to 39° C., after 5 hours of induction, cells were harvested

[0295] Extract Preparation: Cell concentration: .50X . . . in bufferPBS + complete . . . Disruption: press french 3X Centrifugation: 30minutes at 14000 t Comment: >90% in the supernatant of cellular extract

[0296] The cell extract was run on a 12.5% SDS PAGE, and subsequentlystained with Coomassie blue. A Western blot was also performed using ancommercial monoclonal antibody against the poly-histidine tail(Quiagen). The resulting gels (FIGS. 3 and 4), show that the protein isexpressed, and visible in the cell extract supernatant.

[0297] The purification scheme follows a classical approach based on thepresence of an His affinity tail in the recombinant protein. In atypical experiment the disrupted cells are filtered and the acellularextracts loaded onto an Ion Metal Affinity Chromatography (IMAC; Ni++NTAfrom Qiagen) that will specifically retain the recombinant protein.

[0298] The retained proteins are eluted by 0-500 mM imidazole gradient(possibly in presence of a detergent) in a phosphate buffer.

[0299] The supernatant from the harvested culture was denatured in 6Murea, 100 mM NaH₂PO₄, 10 mM Tris, PH 8, and loaded on a chromatographiccolumn IMAC Qiagen NTA Ni⁺⁺ under the following conditions:Equilibration buffer: NaH2Po4 100 mM PH 8 Tris  10 mM Urea  6 M

[0300] Sample:supernatant in urea 6M, 100 mM NaH2Po4, 10 mMTris Washbuffers: 1) NaH2PO4 100 mM PH 8 Tris  10 mM urea  6 M Imidazole  25 mM2) NaH2Po4 100 mM PH 8 Tris  10 mM Urea  6 mM Imidazole  50 mM Elutionbuffer: NaH2PO4 100 mM PH 5.5 Tris  10 mM Urea  6 M Imidazole 500 mM

[0301] The eluted protein in 500 mM imidazole+6M urea is dialysed underthe following conditions:

[0302] PBS PH 7,2+sarkosyl 0.5%+4M urea

[0303] idem at 2M urea 2 hours

[0304] idem at 0M urea 2 hours

[0305] The final material is frozen and stored. The protein content wasquantified using a Lowry protein assay (0.9 mg/1.2 mL). The purity wasassessed by a 12.5% PAGE SDS stained with Coomassie blue (FIG. 5), andthe presence of the recombinant protein was checked by Western blot,using a anti-polyhistidine monoclonal antibody (FIG. 6)

[0306] A comparative evaluation of the different versions of theexpressed antigen will allow the selection of the most promisingcandidate that is to be used for further purification and immunologicalevaluation.

[0307] 9.2 Antibody Production and Immunohistochemistry

[0308] Small amounts of relatively purified protein can be used togenerate immunological tools in order to

[0309] a) detect the expression by immunohistochemistry in normal orcancer tissue sections;

[0310] b) detect the expression, and to follow the protein during thepurification process (ELISA/Western Blot); or

[0311] c) characterize/quantify the purified protein (ELISA).

[0312] 9.2.1 Polyclonal Antibodies:

[0313] Immunization

[0314] Rabbits are immunized, intramuscularly (I.M.), 3 times at 3 weeksintervals with 100 μg of protein, formulated in the adjuvant3D-MPL/QS21. Three weeks after each immunization a blood sample is takenand the antibody titer estimated in the serum by ELISA using the proteinas coating antigen following a standard protocol.

[0315] ELISA

[0316] 96 well microplates (maxisorb Nunc) are coated with 5 μg ofprotein overnight at 4° C. After 1 hour saturation at 37° C. with PBSNCS1%, serial dilution of the rabbit sera is added for 1H 30 at 37° C.(starting at {fraction (1/10)}). After 3 washings in PBS Tween, antirabbit biotinylated anti serum (Amersham ) is added ({fraction(1/5000)}). Plates are washed and peroxydase coupled streptavidin({fraction (1/5000)}) is added for 30 minutes at 37° C. After washing,50 μl TMB (BioRad) is added for 7 minutes and the reaction then stoppedwith H2SO4 0.2M. The OD can be measured at 450 nm and midpoint dilutionscalculated by SoftmaxPro.

[0317] 9.2.2 Monoclonal Antibodies:

[0318] Immunization

[0319] 5 BALB/c mice are immunized 3 times at 3 week intervals with 5 μgof purified protein. Bleedings are performed 14 days post II and 1 weekpost 3. The sera are tested by Elisa on purified protein used as coatedantigen. Based on these results (midpoint dilution >10000) one mouse isselected for fusion.

[0320] Fusion/HAT Selection

[0321] Spleen cells are fused with the SP2/0 myeloma according to astandard protocol using PEG 40% and DMSO 5%. Cells are then seeded in 96well plates 2.5×104-105 cells/well and resistant clones will be selectedin HAT medium. The supernatant of these hybridomas will be tested fortheir content in specific antibodies and when positive, will besubmitted to 2 cycles of limited dilution. After 2 rounds of screening,3 hybridomas will be chosen for ascitis production.

[0322] 9.2.3 Immunohistochemistry

[0323] When antibodies are available, immuno staining is performed onnormal or cancer tissue sections, in order to determine:

[0324] the level of expression of the antigen of the invention in cancerrelative to normal tissue or

[0325] the proportion of cancer of a certain type expressing the antigen

[0326] if other cancer types also express the antigen

[0327] the proportion of cells expressing the antigen in a cancer tissue

[0328] Tissue Sample Preparation

[0329] After dissection, the tissue sample is mounted on a cork disk inOCT compound and rapidly frozen in isopentane previously super cooled inliquid nitrogen (−160° C.). The block will then be conserved at −70° C.until use. 7-10 μm sections will be realised in a cryostat chamber (−20,−30° C.).

[0330] Staining

[0331] Tissue sections are dried for 5 minutes at room temperature(“RT”), fixed in acetone for 10 minutes at RT, dried again, andsaturated with PBS 0.5% BSA 5% serum. After 30 minutes at RT either adirect or indirect staining is performed using antigen specificantibodies. A direct staining leads to a better specificity but a lessintense staining whilst an indirect staining leads to a more intense butless specific staining.

[0332] 9.3 Analysis of Human Cellular Immune Responses to the Antigen ofthe Invention

[0333] The immunological relevance of the antigen of the invention canbe assessed by in vitro priming of human T cells. All T cell lymphocytelines and dendritic cells are derived from PBMCs (peripheral bloodmononuclear cells) of healthy donors (HLA-A2 subtype). An HLA-A2.1/Kbtransgenic mouse model is also used for screening of HLA-A2.1 peptides.

[0334] Newly discovered antigen-specific CD8⁺ T cell lines are raisedand maintained by weekly in vitro stimulation. The lytic activity andthe γ-IFN production of the CD8⁺ lines in response to the antigen orantigen derived-peptides is tested using standard procedures.

[0335] Two strategies to raise the CD8⁺ T cell lines are used: apeptide-based approach and a whole gene-based approach. Both approachesrequire the full-length cDNA of the newly discovered antigen in thecorrect reading frame to be either cloned in an appropriate deliverysystem or to be used to predict the sequence of HLA binding peptides.

[0336] Peptide-Based Approach

[0337] Briefly, transgenic mice are immunized with adjuvanted HLA-A2peptides, those unable to induce a CD8⁺ response (as defined by anefficient lysis of peptide-pulsed autologous spleen cells) will befurther analyzed in the human system.

[0338] Human dendritic cells (cultured according to Romani et al.) willbe pulsed with peptides and used to stimulate CD8⁺-sorted T cells (byFacs). After several weekly stimulations, the CD8⁺ lines will be firsttested on peptide-pulsed autologous BLCL (EBV-B transformed cell lines).To verify the proper in vivo processing of the peptide, the CD8⁺ lineswill be tested on cDNA-transfected tumour cells (HLA-A2 transfectedLnCaP, Skov3 or CAMA tumour cells).

[0339] Whole Gene-Based Approach

[0340] CD8⁺ T cell lines will be primed and stimulated with eithergene-gun transfected dendritic cells, retrovirally transducedB7.1-transfected fibroblasts, recombinant pox virus or adenovirusinfected dendritic cells. Virus infected cells are very efficient topresent antigenic peptides since the antigen is expressed at high levelbut can only be used once to avoid the over-growth of viral T cellslines.

[0341] After alternated stimulations, the CD8⁺ lines are tested oncDNA-transfected tumour cells as indicated above. Peptide specificityand identity is determined to confirm the immunological validation.

[0342] CD4⁺ T-Cell Response

[0343] Similarly, the CD4⁺ T-cell immune response can also be assessed.Generation of specific CD4⁺ T-cells is made using dendritic cells loadedwith recombinant purified protein or peptides to stimulate the T-cells.

[0344] Predicted Epitopes (Nonamers and Decamers) Binding HLA Alleles:

[0345] The HLA Class I binding peptide sequences are predicted either bythe Parker's algorithm (Parker, K. C., M. A. Bednarek, and J. E.coligan. 1994. Scheme for ranking potential HLA-A2 binding peptidesbased on independent binding of individual peptide side-chains. J.Immunol. 152:163 and http://bimas.dcrt.nih.gov/molbio/hla_bind/) or theRammensee method (Rammensee, Friede, Stevanovic, MHC ligands and peptidemotifs: 1st listing, Immunogenetics 41, 178-228, 1995; Rammensee,Bachmann, Stevanovic: MHC ligands and peptide motifs. Landes Bioscience1997, and http://134.2.96.221/scripts/hlaserver.dll/home.htm). Peptidesare then screened in the HLA-A2.1/Kb transgenic mice model (Vitiello etal.).

[0346] The HLA Class II binding peptide sequences are predicted usingthe Tepitope algorithm, with a score cut-off set to 6 (Stumiolo, Hammerat al., Nature Biotechnology. 1999. 17;555-561).

[0347] The following tables gather the Class I and II predicted epitopesequences: Subsequence Start Residue Parker's Rank Position ListingScore° SEQ ID: HLA-A 0201: decamers 1  64 KLVNLGFQAL 142.060 SEQ ID NO:16 HLA-A 0201: nonamers 1 182 ELLDFSSWL 507.976 SEQ ID NO: 17 2 104RLLAEHDAV 126.098 SEQ ID NO: 18 3  64 KLVNLGFQA 100.850 SEQ ID NO: 19Subsequence Start Residue Parker's Rank Position Listing Score SEQ ID:HLA-A 24: nonamers 1  97 EYIRALQRL 360.000 SEQ ID NO: 20 HLA-A 24:decamers 1  97 EYIRALQRLL 360.000 SEQ ID NO: 21 HLA-B 7: decamers 1 111AVRNALAGGL 600.000 SEQ ID NO: 22 HLA-B 4403: decamers 1 156 SEPGSPRSAY360.000 SEQ ID NO: 23 2  89 VETLRSAVEY 180.000 SEQ ID NO: 24 SubsequenceStart Residue Tepitope Rank Position Listing Score SEQ ID:HLA-DRB1*1501: nonamers 1  99 IRALQRLLA 5.6 SEQ ID NO: 25 HLA-DRB1*1502:nonamers 1  99 IRALQRLLA 4.6 SEQ ID NO: 25 1HLA-DRB1*0402: nonamers 1120 LRPQAVRPS 5.4 SEQ ID NO: 26 HLA-DRB1*1101: nonamers 1  99 IRALQRLLA4.8 SEQ ID NO: 25 HLA-DRB1*1102: nonamers 1 120 LRPQAVRPS 6.2 SEQ ID NO:26 HLA-DRB1*1104: nonamers 1  99 IRALQRLLA 5.8 SEQ ID NO: 25HLA-DRB1*1106: nonamers 1  99 IRALQRLLA 5.8 SEQ ID NO: 25 HLA-DRB1*1301:nonamers 1 120 LRPQAVRPS 6.6 SEQ ID NO: 26 2  73 LRQHVPHGG 4.9 SEQ IDNO: 27 3  31 LLRCSRRRR 4.4 SEQ ID NO: 33 HLA-DRB1*1302: nonamers 1 120LRPQAVRPS 5.6 SEQ ID NO: 26 HLA-DRB1*1304: nonamers 1 120 LRPQAVRPS 6.2SEQ ID NO: 26 2  73 LRQHVPHGG 4.8 SEQ ID NO: 27 3  31 LGFQALRQH 4.6 SEQID NO: 28 HLA-DRB1*1305: nonamers 1  99 IRALQRLLA 4.8 SEQ ID NO: 25HLA-DRB1*0703: nonamers 1 112 VRNALAGGL 5.1 SEQ ID NO: 29 2  98YIRALQRLL 4.8 SEQ ID NO: 30 3  65 LVNLGFQAL 4.5 SEQ ID NO: 31HLA-DRB5*0101: nonamers 1 96 VEYIRALQR 4.3 SEQ ID NO: 32

Example 10 CASB7439 Specific Cellular Immune Response

[0348] A further way of assessing CASB7439 immunogenicity is todemonstrate CASB7439 antigen has a potential to trigger a cellularimmune response. For that purpose, it has to be verified the humanCD4+T-cell repertoire has the ability to recognise CASB7439 antigenspresented by APC's (antigen-presenting cells) in a MHC class IIrestricted manner.

[0349] To demonstrate CASB7439 antigen can generate a specificCD4+T-cell activity in human, as well as to identify CASB7439 epitopes,a series of “in vitro priming” experiments have been carried out withthe PBMC's of three healthy donors.

[0350] In Vitro Priming of Donor #1

[0351] In-vitro priming cultures with the PBMC of donor #1 wereestablished using 15-mer peptides overlapping by 11 amino acids from thesequence of CASB7439.

[0352] Peptides were combined in pools of 6 or 7 peptides/pool (theresulting 45 peptide sequences and 7 pools are detailed in table 5), andpulsed onto autologous dendritic cells (DCs). Following 4 stimulationcycles, donor #1 PBMC cell lines were assayed for proliferation by usinga 3H-thymidine incorporation assay and for IFN-γ synthesis by ELISA.

[0353] A number of positive peptide pools were identified, with 21 and 7cells lines exhibited stimulation index (S.I.)>3 and S.I.>5,respectively (Stimulation index reflects the ratio of activity from Tcells incubated with DC pulsed with relevant vs. irrelevant peptide orprotein).

[0354] All positive 21 lines were further re-stimulated with individualpeptides. One line, designated 3H8, showed a specific reactivity topeptide #21.

[0355] To map the particular epitope within peptide 21 and recognised bythe T-cells of donor #1, T-Cell clones ere individualized from theT-cell lines. For this purpose, the line 3H8 was re-stimulated onantigen expanded using polyclonal activator PHA, and cloned on PHA.Clones were then tested for peptide stimulation in IFN-y ELSA assays.

[0356] Several clones from line 3H8 were shown to recognize peptide 21.Clones generated from this 3H8 line recognised peptide but failed torecognise E. coli-derived NS1-CASB7439 protein. Therefore a similar invitro priming procedure with a new donor has been undertaken to generateT-clones able to recognise the whole CASB7439 protein presented byAPC's.

[0357] In Vitro Priming of Donor #2

[0358] In vitro priming experiments were performed with the PBMC from anadditional donor #2 in similar experimental condition as donor #1. Inbrief, PBMC were stimulated with autologous DC pulsed with the 7 poolsof 6-7 peptides at a concentration of 250 ng/ml for each peptide. 29cells lines showed reactivity to pooled peptides, and were furtherassayed on individual peptides (at 250 ng/ml) and on E. coli-derived NS1-CASB7439 protein (10 μg/ml).

[0359] 5 of these lines (lines 3A3, 4C5, 3C9, 4D5, and 4B12)demonstrated specific reactivity to a particular peptide pool and toboth an individual peptide derived from that pool and E. coli-derivedNS1-CASB7439.

[0360] Two of the lines, 3A3 and 4C5, have been cloned using PHA. Theresulting clones were assayed against DCs pulsed with E. coli-derivedNS1-CASB7439 fusion protein (2.5 μg/ml) or with irrelevant protein(OspA: 2.5 μg/ml) and assayed for proliferation (3H-Thy) as well asIFN-γ production. 61 clones from the two cell lines were shown torecognise E. coli-derived protein, and all of the 61 clones have beenalso shown to recognise the peptide 16 from the pool.

[0361] These 16 clones were further characterized by demonstrating MHCClass II restriction. CD4+clones derived from line 3A3 were assayedagainst DCs pulsed with E. coli-derived NS1-CASB7439 protein orirrelevant protein (OspA: 1 μg/ml) in the presence or absence ofantibodies (25 μg/ml) to Class I (W632), Class II (HB 145), HLA-DR(L243), or HLA-DQw3 (HB144). These T cell clones were shown to berestricted by MHC Class II, and more precisely are not DR DQw3restricted. Moreover, a preliminary donor mismatch analyses suggest thatthese clones are likely restricted by HLA-DQ0602.

[0362] Specificity of the NS1-CASB7439 fusion protein generated CD4+ Tcell activity is further demonstrated: indeed, a line 3A3 clone CD4response titers out with CASB7439 protein. No response to OspA, anirrelevant protein used as negative control, is observed.

[0363] In Vitro Priming of Donor #3

[0364] In vitro priming cultures were established from an additionaldonor using the same pools of 15-mer peptide overlapping 11 amino acidsand the same procedures. Four CD4+lines, including 4A7 and 4E4,demonstrated E. coli-derived recombinant protein reactivity that wasblocked by antibody to MHC Class II.

[0365] Furthermore, CD4+ clones derived from lines 4A7 and 4E4 wereassayed against dendritic cells pulsed with E. coli-derived NS1-CASB7439protein or irrelevant protein (OspA: 10 μg/ml), or CASB7439 peptides(250 ng/ml) in the presence or absence of antibodies to Class I (W632:25μg/ml) or anti-HLA-DR (L243:25 μg/ml). CD4+ clones derived from theselines present a specificity that is different from the clones descriedabove, as they are HLA-DR restricted. Moreover, 3 other peptides wereshown to be recognised by CD4+T cells (peptides 23, 24, and 25), thetotal number recognised peptide that were identified being 5 so far.

Example 11 Immunohistochemical Analysis of CASB7439 on Tumour and NormalColon Biopsies

[0366] CASB7439 protein over-expression in colon tumour was verified byimmunohistochemistry (IHC) using a CASB7439 specific rabbit polyclonalantibody (Ab CASB7439 #599, {fraction (1/50)} dilution) directed againstan affinity purified CASB7439 α-peptide (SB599 α-peptide, amino acids 1to 14 of CASB7439).

[0367] Ab CASB7439 #599 was generated as follows: a rabbit is immunizedwith SB599 synthetic α-peptide that is conjugated to a carrier protein(LKH). Conjugate is formulated with Freund's adjuvant, and two rabbitsare immunized with formulated conjugate. Four weeks after the secondimmunization and four weeks after the third immunization, blood samplesare taken. Anti-CASB7439 antibody titers are estimated in the serum byELISA.

[0368] For IHC, paraffin-embedded formalin fixed tissue was sliced into8 micron sections. Steam heat induced epitope retrieval (SHIER) in 0.1 Msodium citrate buffer (pH 6.0) was used for optimal staining conditions.Sections were incubated with 10% serum/PBS for 5 minutes. Tenmicrograms/ml of primary antibody (SB599) was added to each section for25 minutes followed by a 25 minute incubation with a biotinylatedanti-rabbit antibody. Endogenous peroxidase activity was blocked bythree 1.5 minute incubations with hydrogen peroxidase. The avidin biotincomplex/horse radish peroxidase (ABV/HRP) system was used along with DABchromogen to visualise antigen expression. Slides were counterstainedwith hematoxylin.

[0369]FIGS. 7 and 8 shows IHC results on colon tumour #9476 biospy andcolon normal mucosa #9476, respectively. Anti-CASB7439 immunoreactivitywas observed at high level in colon cancer and in normal colon at verylow level. Anti-CASB7439 immunoreactivity was localised to the cytoplasmand associated with the plasma membrane of the cells.

Example 12 Demonstration of CASB7439 in vivo Immunogenicity

[0370] Besides being highly tumour specific, the second criticalcriterion for a candidate vaccine evaluation is its immunogenicity. Oneway of assessing the immunogenicity of a protein is to immunize naiveanimals with synthetic peptides derived from the antigen sequence, andwhich reproduce natural epitopes. The generated anti-peptide antibodieswill then tend to recognise the native antigen, therefore, showing aspecific immune response can be raised against the candidate vaccineantigen.

[0371] Because of the very nature of the immune response, a classicaldose range analysis is replaced by repeated immunizations, allowingcomparison of antibody titers before immunization (control), and aftercumulative injections.

[0372] Two peptides were selected from CASB7439 antigen sequence fortheir immunogenic potential: peptides spanning from amino-acids 1-14(peptide 1) and amino-acids 157-172 (peptide 2). Two rabbits wereimmunized with each peptide, rabbits SB598 and SB599 with peptide 1 andrabbits SB600 and SB601 with peptide 2.

[0373] The selected peptides were conjugated to a carrier protein (KLH).Rabbits were intramuscularly immunized with CASB7439 peptide, 3 times at3 to 4 weeks intervals with 200 μg of conjugate formulated with Freund'sadjuvant. Four weeks after the second immunization (PP) and four weeksafter the third immunization (GP), blood samples were taken, and theCASB7439 specific antibody titers were estimated in the serum by ELISA.Thus, a dose range of 0, 200 and 400 μg of the antigen conjugate isreproduced.

[0374] ELISAs were done in triplicate for each immunizing peptide andrabbit serum, and performed as follows: 96 well microplates were coatedat 4° C. during 16 hours with either 100 ng of CASB7439 peptide or 100ng of KLH as coating antigens. After 2-hour saturation at 25° C. withBSA (1 mg/ml), serial dilution of the rabbit sera was added for 2 hoursat 25° C. (starting at dilution {fraction (1/100)}). Anti rabbitantibody, conjugated with universal-HRP, was then added at dilution{fraction (1/1000)} for 2 hours at 25° C. as secondary antibody. Plateswere washed and OPD (0.4 mg/ml) is added for 30 minutes at 25° C.Reaction was stopped with H2SO44M, and OD measured at 492 nm.

[0375] ELISA results clearly show an antibody immune response againstdistinct CASB7439 synthetic peptide can be raised in several rabbits ina dose-dependant manner. This suggests CASB7439 candidate antigen isindeed immunogenic and, when properly formulated with adjuvant as avaccine, CASB7439 vaccine is able to induce a strong and specificantibody immune response.

Example 13 Real-Time RT-PCR Analysis in a Panel of Tumour and MatchedNormal Tissues of Breast from Multiple Patient Samples

[0376] Real-time RT-PCR (U. Gibson. 1996. Genome Research: 6,996) isused to compare mRNA transcript abundance of the candidate antigen in apanel of tumour and matched normal tissues of breast from multiplepatient samples. In addition, mRNA levels of the candidate antigen indifferent normal and tumour cell lines of breast, in a panel of normaltissues, and finally in breast tumours grown in vivo in SCID mice arealso evaluated by this approach. This analysis establishes the breasttumour specificity of CASB7439 expression, which is an importantcriterion that a good antigen candidate must fulfil.

[0377] cDNA was derived from prostate, ovarian, colon and breast tumourcell lines as well as breast tumours, normal breast and other normaltissues. Human tumour explants from breast cancer patients (˜1 mm³) areimplanted subcutaneously in SCID mice. Time-release hormone pellets(β-estradiol) are co-implanted subcutaneously. When tumours reach ˜5% ofthe total mouse body weight they are excised and a portion (˜1 mm³) ispassaged into another set of SCID mice. This process can be repeatedindefinitely. SCID-derived tumour specimens are designated by thepassage number followed by ‘p’. Real time PCR analysis was performed onmatched pairs (early passage/late passage) of SCID-derived human tumours

[0378] Total RNA is extracted from snap frozen biopsie or cell linesusing TriPure reagent (Boehringer). Poly-A+ mRNA is purified from totalRNA after DNAase treatment using oligo-dT magnetic beads (Dynal).Quantification of the mRNA is performed by spectrofluorimetry(VersaFluor, BioRad) using SybrII dye (Molecular Probes).

[0379] Primers for real-time PCR amplification are designed with thePerkin-Elmer Primer Express software using default options for TaqManamplification conditions. Real time PCR was performed with CASB7439specific probes and the expression levels in the tissues determined: 2distinct TaqMan primer pairs are designed (one pair in CASB7439 3′ UTRregion and one pair in CASB7439 ORF region) and used in subsequentreal-time RT-PCR analysis.

[0380] Real-time reactions are assembled according to standard PCRprotocols using 2 ng of reverse transcribed mRNA (Expand RT, Roche) foreach reaction. Sybrl dye (Molecular Probes) is added at a final dilutionof {fraction (1/75000)} for real-time detection. Amplification (40cycles) and real-time detection is performed in a Perkin-ElmerBiosystems PE7700 system using conventional instrument settings. Ctvalues are calculated using the PE7700 Sequence Detector software, andresults are standardised with respect to Actin.

[0381]FIG. 9 demonstrates while almost all colon cell lines expressedCASB7439 so did the breast tumour cell lines MCF-7, MDA-MB415, BT474 andT47D. These results on a panel of cell lines suggest that, in additionto colon cancer, CASB7439 transcript is also surprisingly over-expressedin breast tumour cell lines, as compared to other normal tissues.Expression is also seen in pooled breast tumour tissue, SCID-derivedbreast tumour 9987 (both passages) and 9077 (late passage only), asshown in FIGS. 10 and 11. Primers derived from the CASB7439 ORF as wellas the 3′ end yielded similar profiles. FIGS. 12-14 confirms breasttumour specificity of CASB7439 expression on an extended panel of breasttumours and normal tissues. Taken together, these real-time RT-PCRresults clearly indicate CASB7439 is over-expressed in an majority ofbreast tumours, as compared to normal tissues.

Example 14 Expression and Purification of Tumour-Specific Antigens

[0382] 14.1 Expression of the Present Invention Antigen in MicrobialHosts.

[0383] Expression in microbial hosts, or alternatively in vitrotranscription/translation, is used to produce the antigen of theinvention for vaccine purposes and to produce protein fragments or wholeprotein for rapid purification and generation of antibodies needed forcharacterization of the naturally expressed protein by western blot,immunohistochemistry or for follow-up of purification.

[0384] Recombinant proteins may be expressed in two microbial hosts, E.coli and in yeast (such as Saccharomyces cerevisiae or Pichia pastoris).The use of two expression hosts allows the selection of the expressionsystem with the best features for this particular antigen production. Ingeneral, the recombinant antigen will be expressed in E. coli and thereagent protein expressed in yeast.

[0385] The expression strategy first involves the design of the primarystructure of the recombinant antigen. In general, an expression fusionpartner (EFP) is placed at the N terminal extremity to improve levels ofexpression that could also include a region useful for modulating theimmunogenic properties of the antigen, an immune fusion partner (IFP).In addition, an affinity fusion partner (AFP) useful for facilitatingfurther purification is included at the C-terminal end. Moreover,polynucleotide sequence of the coding region of the antigen can also bemodified to optimise its expression. Using a codon usage that is adaptedto the chosen recombinant expression host can dramatically increaseantigen expression yields.

[0386] A comparative evaluation of the different versions of theexpressed antigen will allow the selection of the most promisingcandidate that is to be used for further purification and immunologicalevaluation.

[0387] Several constructs underwent comparative evaluation. For rapidexpression and purification as well as generation of antibodies againstCASB7439, it is proposed to generate in E. coli a full length CASB7439protein with Influenza protein NS1 as EFP and a histidine tail as AFP.

[0388] Five constructs have been designed:

[0389] Construct 1: Full length wild type CASB7439 cDNA in fusion withthe N terminal fragment (the first 80 amino acids) of Influenza proteinNS1 coding cDNA as EFP and with a histidine tail coding cDNA as an AFP(SEQ ID NO:8). The encoded fusion protein sequence is SEQ ID NO:10. TheCASB7439 protein design is shown in FIG. 15.

[0390] Construct 2: Full length mutated CASB7439 cDNA in fusion with theN terminal fragment of Influenza protein NS1 coding cDNA as EFP and witha histidine tail coding cDNA as an AFP (SEQ ID NO:9). This construct wasdesigned in order to comprise the first 50 codons of native CASB7439cDNA replaced by codons specific of the E. coli codon usage, to enhanceexpression potential of CASB7439 in its E. coli host. Only CASB7439codons that are known to be problematic in E. coli host by the manskilled in the art were replaced by an E. coli codon. For each CASB7439problematic codon, the E. coli codon is chosen to encode for the sameaminoacid and to have a low GC content. The encoded fusion proteinsequence is SEQ ID NO:10.

[0391] Construct 3: Full length wild type CASB7439 cDNA in fusion with a11 amino acids T7·Tag® (Novagen, Madison, Wis.) coding cDNA as EFP andwith a histidine tail coding cDNA as an AFP (SEQ ID NO:34). The encodedfusion protein is SEQ ID NO:35.

[0392] Construct 4: Full length mutated CASB7439 cDNA in fusion with a11 amino acids T7·Tage® (Novagen, Madison, Wis.) coding cDNA as EFP andwith a histidine tail coding cDNA as an AFP (SEQ ID NO: 36). The first50 codons of native CASB7439 cDNA replaced by codons specific of the E.coli codon usage.The same substitutions as those performed in construct2 have been carried out in construct 4. The encoded fusion proteinsequence is SEQ ID NO:35.

[0393] Construct 5: Full length mutated CASB7439 cDNA is fusion with a11 amino acids T7·Tag® (Novagen, Madison, Wis.) coding cDNA as EFP and ahistine tail coding cDNA as AFP (SEQ ID NO:37). This construct also hasthe first 50 codons of native CASB7439 cDNA replaced by codons specificof the E. coli codon usage. All CASB7439 codons are modified in thisconstruct to have specific E. coli codons and to respect E. coli codonfrequency distribution. The encoded fusion protein sequence is SEQ IDNO:35.

[0394] Tables of E. coli codon usage used for codon optimisation for theabove-mentioned constructs is given in “Codon usage in regulatory genesin E. coli does nor reflect selection for rare codons”, Sharp P. M.,Wen-Hsiung L., Nucleic Acids Research, 14, 7737-7749 (1986).

[0395] Constructs 1 and 2 are cloned in pMG1 plasmid. Plamid pMG1 is aderivative of pMG27N. Plasmid pMG27N is a pBR322-derived expressionvector which contains the P_(L) promoter, an N utilization site (torelieve transcriptional polarity in the presence of N protein) and thecII ribisome binding site including the cII translation initiation codonincorporated in an Nde I restriction site (Gross et al. 1985, Mol. &Cell. Biol. 5:1015). Plasmid pMG1 has been constructed by inserting the81 first codons of the NS1 coding region from influenza strain A/PR/8/34cleaved from plasmid pAS1ΔEH/801 (Young et al. 1983. Proc. Natl. Acad.Sci. USA 80:6105) by BamH I and Nco I into pMG27N digested by BamH I andSac I. A synthetic DNA linker was introduced between the Nco I and theSac I sites. A schematic representation of pMG1 construction is shown inFIG. 16.

[0396] Construct 3 to 5 are cloned in a commercial pET24b(+) plasmidfrom Novagen (Madison, Wis.).

[0397] Constructs 1 to 5 are transformed in the AR58 E. coli strain,which is a cryptic λ lysogen derived from N99 that is gal E::Tn10,Δ-8(chlD-pgl),Δ-H1(cro-chlA),N⁺, and cI857 (Proc. Natl. Acad. Sci.USA vol82, pp.88-92, January 1985 Biochemistry).

[0398] When the recombinant AR58 strains are available, the recombinantproduct is characterized by the evaluation of the level of expressionand the prediction of further solubility of the protein by analysis ofthe behavior in the crude extract. After growth on appropriate culturemedium and induction of the recombinant protein expression, totalextracts are analyzed by SDS-PAGE. The recombinant proteins arevisualised in stained gels and identified by Western blot analysis usingspecific antibodies.

[0399] Expression of the Recombinant Protein from Constructs 1 to 5:

[0400] Bacteria was grown in LB medium+50 μg/ml Kan at 30° C. When theculture reached OD=0.5 (620 nm), the culture was heated up to 39° C.,after 5 hours of induction by ITPG, cells were harvested. The cellextract was prepared as follows: cells were resuspended in PBS buffer,disrupted (French press, 3 times), and centrifuged 30 minutes at 14000t. More than 90% of the protein was found in the supernatant of the cellextract.

[0401] Purification of the Recombinant Protein from Constructs 1 to 5.

[0402] The purification scheme follows a classical approach based on thepresence of an His affinity tail in the recombinant protein. In atypical experiment, the disrupted cells are filtered and the acellularextracts loaded onto an Ion Metal Affinity Chromatography (IMAC; Ni⁺⁺NTAfrom Qiagen) that will specifically retain the recombinant protein. Theretained proteins are eluted by 0-500 mM imidazole gradient (possibly inpresence of a detergent) in a phosphate buffer.

[0403] The supernatant from the harvested culture was denatured in 6Murea, 100 mM NaH₂PO₄, 10 mM Tris, pH 8, and loaded on a chromatographiccolumn IMAC Qiagen NTA Ni⁺⁺ under the following conditions:

[0404] Equilibration buffer:NaH2Po4 100 mM (PH 8) Tris 10 mM Urea6M.

[0405] Sample: supernatant in urea 6M, 100 mM NaH2Po4, 10 mMTris

[0406] Wash buffers:

[0407] 1) NaH2PO4 100 mM PH 8Tris 10 mM Urea 6 M Imidazole 25 mM

[0408] 2) NaH2Po4 100 mM (PH8) Tris 10 mM Urea 6mM Imidazole 50 mM

[0409] Elution buffer: NaH2PO4 100 mM (PH 5,5) Tris 10 mM Urea 6MImidazole 500 mM

[0410] The eluted protein in 500 mM imidazole+6M urea is dialysed underthe following conditions:

[0411] 1. PBS PH 7,2+sarkosyl 0.5%+4M Urea

[0412] 2. idem at 2M Urea 2 hours

[0413] 3. idem at 0M Urea 2 hours

[0414] Recombinant Expression in Microbial Hosts Results:

[0415] Construct 1 (NS1-CASB7439-HIS fusion protein):

[0416] The unpurified cell extracts is run on a 12.5% SDS PAGE, andsubsequently stained with Coomassie blue. A Western blot is alsoperformed using a monoclonal antibody directed against the 80 aminoacidsN terminal fragment of the NS1 protein in fusion with CASB7439construct 1. The resulting gels (FIGS. 17 and 18) show that the proteinis expressed and visible in the cell extract supernatant.

[0417] The final purified material is frozen and stored. The proteincontent was quantified using a Lowry protein assay (0.9 mg/1.2 ml). Thepurity was assessed by a 12.5% PAGE SDS stained with Coomassie blue, andthe presence of the recombinant protein was checked by Western blot,using a monoclonal antibody directed against the 80 aminoacids Nterminal fragment of the NS1 protein in fusion with CASB7439 construct 1(FIG. 19).

[0418] Constructs 3 to 5 (T7 Tag—CASB7439—HIS Fusion Protein):

[0419] Several dilutions of 15 μl of purified material for eachconstruction is run on a 12.5% PAGE SDS gel. Presence andsemi-quantification of recombinant protein is done by Western blot usingSB600 anti-peptide antibody (see section 6.2.2 below for antibodydescription). Results, shown in FIG. 20, clearly indicate CASB7439constructs that have optimized codons for their E. coli host (constructs3 and 4) yield higher quantities of recombinant protein. Using modifiedversion of CASB7439 cDNAs therefore increases CASB7439 recombinantexpression yields.

[0420] 14.2 Expression of the Present Invention Antigen in VaccinationVectors

[0421] The immunogenicity of the antigen of the present invention hasbeen verified by immunizing rabbits and mice using various means ofimmunization. Indeed, immunization with CASB7439 forms, either peptideor recombinant protein could induce humoral immune response with thegeneration of specific antibodies against CASB7439 and/or could induce aCASB7439 specific cellular immune response. Additionally, in vivodelivery of CASB7439 protein using for instance, naked DNA in anappropriate vector encoding CASB7439 or fragments of CASB7439, CASB7439gene delivered by a viral vector encoding CASB7439 or fragments ofCASB7439, is equally appropriate to demonstrate CASB7439 immunogenicity.

[0422] Recombinant Expression of CASB7439 in DNA Vaccination Vectors

[0423] Several vectors can be used to construct the vaccinating plasmid(for instance, pcDNA3.1 vector, pJB16 vector). The plasmid expressionvector for CASB7439 (pJB76) was constructed through the recombination ofpDOR207/CASB7439 with the destination vector pJB16 (pVR1012-Dest) by theLR reaction of the Gateway Cloning Technology. HEK293T cells weretransfected in 6-well plate with pJB76 DNA (0.25 or 1 μg), or emptyvector (pJB16; 1 ug). Cells were harvested at 48 hours post-infectionand cell lysates were analyzed by Western blot for expression ofCASB7439 using rabbit anti-NS1-CASB7439-His polyclonal antibody(dilution 1:500, see section 6.2.1 for antibody description). As shownin FIG. 21, a band at the size expected for CASB7439 is observed at lowlevel in fibroblasts transfected with 1 ug of CASB7439 DNA (pJB76). Noband is observed using with empty vector (pJB16).

[0424] Recombinant Expression of CASB7439 in a Adenoviral Live Vector

[0425] Recombinant adenoviruses are effective vectors for gene-basedvaccination because they are capable of eliciting humoral and cellularimmune responses against the encoded antigen. Recombining CASB7439 bysuch an approach is recommended for further immunological validations.Recombinant adenovirus for CASB7439 (AdC7439) was constructed using aLac/GW-1 expression cassette under control of the CMV promoter.

[0426] A schematic representation of the recombinant expression vectoris shown in FIG. 22. In brief, 2×10⁵ human fibroblasts from donor D93were infected by the recombinant adenovirus for CASB7439 (AdC7439) in6-well plate at various MOI. Cells were harvested at 48 hourspost-infection and cell lysates were analyzed by Western blot forexpression of CASB7439 using rabbit anti-NS1-CASB7439-His polyclonalantibody (1:500). As shown in FIG. 23, a band at the size expected forCASB7439 is observed at low level in fibroblasts infected at an MOI of200 and 300 and at high level at MOI of 400 or 500. No expression isseen in uninfected fibroblasts.

Example 15 Real-Time RT-PCR Analysis of the Candidate Antigen in Panelsof Tumour and Preneoplasic Tissues of Lung

[0427] Real-time RT-PCR (U. Gibson. 1996. Genome Research: 6,996) isused to evaluate and compare mRNA transcript abundance of the candidateantigen in panels of tumour and preneoplasic tissues of lung frommultiple patient samples, of normal tissues, of different normal andtumour cell lines of lung, and finally of lung tumours grown in vivo inSCID mice. This analysis is important to establish the lung tumourspecificity of CASB7439 expression, which is an important criterion agood antigen candidate must fulfil.

[0428] Primers for real-time PCR amplification are designed with thePerkin-Elmer Primer Express software using default options for TaqManamplification conditions.

[0429] Human tumour explants from lung cancer patients are implantedsubcutaneously in SCID mice. When tumours reach ˜5% of the total mousebody weight they are excised and a portion (˜1 mm³) is passaged intoanother set of SCID mice. This process can be repeated indefinitely.Real time PCR analysis was performed on matched pairs (earlypassage/late passage) of SCID-derived human tumours.

[0430] Total RNA is extracted using TriPure reagent (Boehringer) andDNAse treatment from snap frozen biopsies of preneoplasic lesions oflung, early stage (stages Ib or IIa or IIb) non small cell lungcarcinoma (NSCLC), late stage (stages IlIa or IIIb or IV) NSCLC, andseveral normal tissues, or from normal and tumour cell lines or lungtumours grown in vivo in SCID mice. Quantification of total RNA isperformed by spectrofluorimetry (VersaFluor, BioRad) using SybrII dye(Molecular Probes), and cDNA is obtained from the quantified total RNAby reverse transcription using a poly-dT oligonucleotide and Expand(Roche) reverse transcriptase.

[0431] Real-time reactions are assembled according to standard PCRprotocols using 2 ng of reverse transcribed mRNA for each reaction.SybrI dye (Molecular Probes) is added at a final dilution of {fraction(1/75000)} for real-time detection. Amplification (40 cycles) andreal-time detection is performed in a Perkin-Elmer Biosystems PE7700system using conventional instrument settings. Ct values are calculatedusing the PE7700 Sequence Detector software. Several Ct values areobtained for each samples: for the panel of normal tissue samples, aCtXY for each normal tissue XY is calculated. An another Ct (CtA) isalso obtained on β-Actin gene, as an internal reference, for all of thesamples.

[0432] As the efficiency of PCR amplification under the prevailingexperimental conditions is close to the theoretical amplificationefficiency, 2(CtA-CtXY) value is an estimate of the relative TAAtranscript level of the sample, standardised with respect to Actintranscript level. A value of 1 thus suggests the candidate antigen andActin have the same expression level.

[0433] Absolute levels of CASB7439 transcript, as assessed byquantitative RT-PCR, in preneoplasic lesions of lung, early and latestage lung cancers and normal tissues is shown in FIGS. 20 to 28.Comparison of CASB7439 transcript abundance in preneoplasic lesions oflung, early and late stage lung cancers to normal tissues is depicted intable 1. This clearly show CASB7439 transcript is over-expressed in avast majority of patients suffering from lung preneoplasic lesions andlate stage lung cancer. Overexpression fold in both populations ofpatients is close to 5. Some overexpression at comparable levels isindeed also seen in an early stage lung cancer population of patients,but coverage of overexpression is more restricted than those ofpreneoplasic lesions and late stage lung cancer populations (see Table6). In conclusion, CASB7439 lung preneoplasic lesion and lung tumourspecificity agrees with its use a diagnostic and immunotherapeutic agentin a lung cancer context. TABLE 6 CASB7439 transcript overexpressioncoverages and levels in lung preneoplasic, early and late stage lungcancer patient populations as compared to several normal tissues. % ofpreneoplasic patients showing a CASB7439 overexpression 100%  Averageoverexpression fold in overexpressing preneoplasic 5.04 patients Medianoverexpression fold in overexpressing preneoplasic 4.78 patients % ofearly stage lung cancer patients showing a CASB7439 33% overexpressionAverage overexpression fold in overexpressing early stage lung 4.47cancer patients Median overexpression fold in overexpressing early stagelung 2.87 cancer patients % of late stage lung cancer patients showing aCASB7439 83% overexpression Average overexpression fold inoverexpressing late stage lung 4.69 cancer patients Medianoverexpression fold in overexpressing late stage lung 3.21 cancerpatients

Example 16 Northern-Southern Blot Analysis

[0434] Limited amounts of mixed tumour and normal lung cDNA areamplified by Advantage PCR (see above). Messenger RNA from multiplenormal tissues is also amplified using the same procedure. The amplifiedcDNA (1 μg) is electrophoresed on a 1.2% agarose gel and transferredonto a nylon membrane. The membrane is hybridised (AlkPhos DirectSystem) with a probe prepared using a fragment of the candidate TAAcDNA. Northern-Southern analysis provides information on transcriptsize, presence of splice variants and transcript abundance in tumour andnormal tissues.

Example 17 17.1 Expression and Purification of Tumour-Specific Antigens

[0435] 17.1.1 Expression of the Present Invention Antigen in MicrobialHosts.

[0436] Expression in microbial hosts, or alternatively in vitrotranscription/translation, is used to produce the antigen of theinvention for vaccine purposes and to produce protein fragments or wholeprotein for rapid purification and generation of antibodies needed forcharacterization of the naturally expressed protein by western blot,immunohistochemistry or for follow-up of purification.

[0437] Recombinant proteins may be expressed in two microbial hosts, E.coli and in yeast (such as Saccharomyces cerevisiae or Pichia pastoris).This allows the selection of the expression system with the bestfeatures for this particular antigen production. In general, therecombinant antigen will be expressed in E. coli and the reagent proteinexpressed in yeast.

[0438] The expression strategy first involves the design of the primarystructure of the recombinant antigen. In general an expression fusionpartner (EFP) is placed at the N terminal extremity to improve levels ofexpression that could also include a region useful for modulating theimmunogenic properties of the antigen, an immune fusion partner (IFP).In addition, an affinity fusion partner (AFP) useful for facilitatingfurther purification is included at the C-terminal end. Moreover,polynucleotide sequence of the coding region of the antigen can also bemodified to optimise its expression. Using a codon usage that is adaptedto the chosen recombinant expression host can dramatically increaseantigen expression yields.

[0439] A comparative evaluation of the different versions of theexpressed antigen will allow the selection of the most promisingcandidate that is to be used for further purification and immunologicalevaluation.

[0440] Several constructs underwent comparative evaluation: For rapidexpression and purification as well as generation of antibodies againstCASB7439, it is proposed to generate in E. coli a full length CASB7439protein with Influenza protein NS1 as EFP and a histidine tail as AFP.

[0441] Five constructs have been designed as presented in Example 14:

[0442] Construct 1: Full length wild type CASB7439 cDNA in fusion withthe N terminal fragment (the first 80 amino acids) of Influenza proteinNS1 coding cDNA as EFP and with a histidine tail coding cDNA as an AFP(SEQ ID NO:8). The encoded fusion protein sequence is SEQ ID NO:10. TheCASB7439 protein design is shown in FIG. 29.

[0443] Contruct 2: Full length mutated CASB7439 cDNA in fusion with theN terminal fragment of Influenza protein NS1 coding cDNA as EFP and witha histidine tail coding cDNA as an AFP (SEQ ID NO:9). This construct wasdesigned in order to comprise the first 50 codons of native CASB7439cDNA replaced by codons specific of the E. coli codon usage, to enhanceexpression potential of CASB7439 in its E. coli host. Only CASB7439codons that are known to be problematic in E. coli host by the manskilled in the art were replaced by an E. coli codon. For each CASB7439problematic codon, the E. coli codon is chosen to encode for the sameamino acid and to have a low GC content. The encoded fusion proteinsequence is SEQ ID NO:10.

[0444] Contruct 3: Full length wild type CASB7439 cDNA in fusion with a11 amino acids T7·Tag® (Novagen, Madison, Wis.) coding cDNA as EFP andwith a histidine tail coding cDNA as an AFP (SEQ ID NO:34). The encodedfusion protein is SEQ ID NO:35.

[0445] Construct 4: Full length mutated CASB7439 cDNA in fusion with a11 amino acids T7·Tag® (Novagen, Madison, Wis.) coding cDNA as EFP andwith a histidine tail coding cDNA as an AFP (SEQ ID NO:36). The first 50codons of native CASB7439 cDNA replaced by codons specific of the E.coli codon usage.The same substitutions as those performed in construct2 have been carried out in Contruct 4. The encoded fusion proteinsequence is SEQ ID NO:35.

[0446] Construct 5: Full length mutated CASB7439 cDNA is fusion with a11 amino acids T7·Tag® (Novagen, Madison, Wis.) coding cDNA as EFP and ahistine tail coding cDNA as AFP (SEQ ID NO:37). This construct also hasthe first 50 codons of native CASB7439 cDNA replaced by codons specificof the E. coli codon usage. All CASB7439 codons are modified in thisconstruct to have specific E. coli codons and to respect E. coli codonfrequency distribution. The encoded fusion protein sequence is SEQ IDNO:35.

[0447] Tables of E. coli codon usage used for codon optimisation for theabove-mentioned constructs is given in “Codon usage in regulatory genesin E. coli does nor reflect selection for rare codons”, Sharp P. M.,Wen-Hsiung L., Nucleic Acids Research, 14, 7737-7749 (1986).

[0448] Constructs 1 and 2 are cloned in pMG1 plasmid. Plasmid pMG1 is aderivative of pMG27N. Plasmid pMG27N is a pBR322-derived expressionvector which contains the PL promoter, an N utilization site (to relievetranscriptional polarity in the presence of N protein) and the cIIribisome binding site including the cII translation initiation codonincorporated in an Nde I restriction site (Gross et al. 1985, Mol. &Cell. Biol. 5:1015). Plasmid pMG1 has been constructed by inserting the81 first codons of the NS1 coding region from influenza strain A/PR/8/34cleaved from plasmid pAS1ΔEH/801 (Young et al. 1983. Proc. Natl. Acad.Sci. USA 80:6105) by BamH I and Nco I into pMG27N digested by BamH I andSac I. A synthetic DNA linker was introduced between the Nco I and theSac I sites. A schematic representation of pMG1 construction is shown inFIG. 30.

[0449] Construct 3 to 5 are cloned in a commercial pET24b(+) plasmidfrom Novagen (Madison, Wis.).

[0450] Constructs 1 to 5 are transformed in the AR58 E. coli strain,which is a cryptic λ lysogen derived from N99 that is gal E::Tn10,Δ-8(chlD-pgl),Δ-H1(cro-chlA),N+, and cI857 (Proc. Natl. Acad. Sci.USA vol82, pp.88-92, January 1985 Biochemistry).

[0451] When the recombinant AR58 strains are available, the recombinantproduct is characterized by the evaluation of the level of expressionand the prediction of further solubility of the protein by analysis ofthe behavior in the crude extract. After growth on appropriate culturemedium and induction of the recombinant protein expression, totalextracts are analyzed by SDS-PAGE. The recombinant proteins arevisualised in stained gels and identified by Western blot analysis usingspecific antibodies.

[0452] Expression of the Recombinant Protein from Constructs 1 to 5:

[0453] Bacteria was grown in LB medium+50 μg/ml Kan at 30° C. When theculture reached OD=0.5 (620 nm), the culture was heated up to 39° C.,after 5 hours of induction by ITPG, cells were harvested. The cellextract was prepared as follows: cells were resuspended in PBS buffer,disrupted (French press, 3 times), and centrifuged 30 minutes at 14000t. More than 90% of the protein was found in the supernatant of the cellextract.

[0454] Purification of the Recombinant Protein from Constructs 1 to 5.

[0455] The purification scheme follows a classical approach based on thepresence of an His affinity tail in the recombinant protein. In atypical experiment the disrupted cells are filtered and the acellularextracts loaded onto an Ion Metal Affinity Chromatography (IMAC; Ni++NTAfrom Qiagen) that will specifically retain the recombinant protein.

[0456] The retained proteins are eluted by 0-500 mM imidazole gradient(possibly in presence of a detergent) in a phosphate buffer.

[0457] The supernatant from the harvested culture was denatured in 6Murea, 100 mM NaH2PO4, 10 mM Tris, pH 8, and loaded on a chromatographiccolumn IMAC Qiagen NTA Ni++ under the following conditions:

[0458] Equilibration buffer:NaH2Po4 100 mM (PH 8) Tris 10 mM Urea6M.

[0459] Sample: supernatant in urea 6M, 100 mM NaH2Po4, 10 mMTris

[0460] Wash buffers:

[0461] 1) NaH2PO4 100 mM PH 8Tris 10 mM Urea 6 M Imidazole 25 mM

[0462] 2) NaH2Po4 100 mM (PH8) Tris 10 mM Urea 6mM Imidazole 50 mM

[0463] Elution buffer: NaH2PO4 100 mM (PH 5,5) Tris 10 mM Urea 6MImidazole 500 mM

[0464] The eluted protein in 500 mM imidazole+6M urea is dialysed underthe following conditions:

[0465] PBS PH 7,2+sarkosyl 0.5%+4M Urea

[0466] idem at 2M Urea 2 hours

[0467] idem at 0M Urea 2 hours

[0468] Recombinant Expression in Microbial Hosts Results:

[0469] Construct 1 (NS1-CASB7439-HIS Fusion Protein):

[0470] The unpurified cell extracts is run on a 12.5% SDS PAGE, andsubsequently stained with Coomassie blue. A Western blot is alsoperformed using a monoclonal antibody directed against the 80 aminoacidsN terminal fragment of the NS1 protein in fusion with CASB7439construct 1. The resulting gels (FIGS. 17 and 18) show that the proteinis expressed and visible in the cell extract supernatant.

[0471] The final purified material is frozen and stored. The proteincontent was quantified using a Lowry protein assay (0.9 mg/1.2 mL). Thepurity was assessed by a 12.5% PAGE SDS stained with Coomassie blue, andthe presence of the recombinant protein was checked by Western blot,using a monoclonal antibody directed against the 80 aminoacids Nterminal fragment of the NS1 protein in fusion with CASB7439 construct 1(FIG. 19).

[0472] Constructs 3 to 5 (T7 Tag—CASB7439—HIS Fusion Protein):

[0473] Several dilutions of 15 μl of purified material for eachconstruction is run on a 12.5% PAGE SDS gel. Presence andsemi-quantification of recombinant protein is done by Western blot usingSB600 anti-peptide antibody (see section 17.2.2 below for antibodydescription). Results, shown in FIG. 31, clearly indicate CASB7439constructs that have optimized codons for their E. coli host (constructs3 and 4) yield higher quantities of recombinant protein. Using modifiedversion of CASB7439 cDNAs therefore increases CASB7439 recombinantexpression yields.

[0474] 17.1.2 Expression of the Present Invention Antigen in VaccinationVectors

[0475] The immunogenicity of the antigen of the present invention hasbeen verified by immunizing rabbits and mice using various means ofimmunization. Indeed, immunization with CASB7439 forms, either peptideor recombinant protein could induce humoral immune response with thegeneration of specific antibodies against CASB7439 and/or could induce aCASB7439 specific cellular immune response. Additionally, in vivodelivery of CASB7439 protein using for instance, naked DNA in anappropriate vector encoding CASB7439 or fragments of CASB7439, CASB7439gene delivered by a viral vector encoding CASB7439 or fragments ofCASB7439, is equally appropriate to demonstrate CASB7439 immunogenicity.

[0476] Recombinant Expression of CASB7439 in DNA Vaccination Vectors

[0477] Several vectors can be used to construct the vaccinating plasmid(for instance, pcDNA3.1 vector, pJB16 vector). The plasmid expressionvector for CASB7439 is constructed through the recombination of aCASB7439 recombinant plasmid with a destination vaccinating plasmid suchas pcDNA3.1 or pJB 16 by the LR reaction of the Gateway CloningTechnology. HEK293T cells are transfected in 6-well plate withrecombinant vaccinating plasmid DNA (0.25 or 1 μg), or empty vector (1μg). Cells were harvested at 48 hours post-infection and cell lysateswere analyzed by Western blot for expression of CASB7439 using rabbitanti-NS1-CASB7439-His polyclonal antibody (dilution 1:500, see section6.2.1 for antibody description).

[0478] Recombinant Expression of CASB7439 in a Adenoviral Live Vector

[0479] Recombinant adenoviruses are effective vectors for gene-basedvaccination because they are capable of eliciting humoral and cellularimmune responses against the encoded antigen. Recombining CASB7439 bysuch an approach is recommended for further immunological validations.Recombinant adenovirus for CASB7439 was constructed using a Lac/GW-1expression cassette under control of the CMV promoter. In brief, 2×10⁵human fibroblasts were infected by the CASB7439 recombinant adenovirusin 6-well plate at various MOI. Cells were harvested at 48 hourspost-infection and cell lysates were analyzed by Western blot forexpression of CASB7439 using rabbit anti-NS1-CASB7439-His polyclonalantibody (1:500).

[0480] 17.2 Antibody Production and Immunohistochemistry

[0481] Small amounts of relatively purified protein can be used togenerate immunological tools in order to

[0482] a) detect the expression by immunohistochemistry in normal orcancer tissue sections;

[0483] b) detect the expression, and to follow the protein during thepurification process (ELISA/Western Blot); or

[0484] c) characterize/quantify the purified protein (ELISA).

[0485] 17.2.1 Polyclonal Antibodies:

[0486] Immunisation

[0487] Rabbits were immunized, intramuscularly (I.M.), 3 times at 3weeks intervals with 100 μg of protein, formulated in the adjuvant3D-MPL/QS21. Three weeks after each immunization a blood sample wastaken and the antibody titer estimated in the serum by ELISA using theprotein as coating antigen following a standard protocol.

[0488] ELISA

[0489] 96 well microplates (maxisorb Nunc) were coated with 5 μg ofprotein overnight at 4° C. After 1 hour saturation at 37° C. with PBSNCS 1%, serial dilution of the rabbit sera is added for 1H 30 at 37° C.(starting at {fraction (1/10)}). After 3 washings in PBS Tween, antirabbit biotinylated anti serum (Amersham ) is added ({fraction(1/5000)}). Plates are washed and peroxydase coupled streptavidin({fraction (1/5000)}) is added for 30 minutes at 37° C. After washing,50 μl TMB (BioRad) is added for 7 minutes and the reaction then stoppedwith H₂SO₄ 0.2M. The OD can be measured at 450 nm and midpoint dilutionscalculated by SoftmaxPro.

[0490] 17.2.2 Anti-Peptide Antibodies.

[0491] Besides being highly tumour specific, the second criticalcriterion for a candidate vaccine evaluation is its immunogenicity. Oneway of assessing the immunogenicity of a protein is to immunize naïveanimals with synthetic peptides derived from the antigen sequence, andwhich reproduce natural epitopes. The generated anti-peptide antibodieswill then tend to recognise the native antigen, therefore showing aspecific immune response can be raised against the candidate vaccineantigen.

[0492] Because of the very nature of the immune response, a classicaldose range analysis is replaced by repeated immunizations, allowingcomparison of antibody titers before immunization (control), and aftercumulative injections.

[0493] Two peptides were selected from CASB7439 antigen sequence fortheir immunogenic potential: peptides spanning from amino-acids 1-14(peptide 1) and amino-acids 157-172 (peptide 2). Two rabbits wereimmunized with each peptide, rabbits SB598 and SB599 with peptide 1 andrabbits SB600 and SB601 with peptide 2.

[0494] The selected peptides were conjugated to a carrier protein (KLH).Rabbits were intramuscularly immunized with CASB7439 peptide, 3 times at3 to 4 weeks intervals with 200 μg of conjugate formulated with Freund'sadjuvant. Four weeks after the second immunization (PP) and four weeksafter the third immunization (GP), blood samples were taken, and theCASB7439 specific antibody titers were estimated in the serum by ELISA.Thus, a dose range of 0, 200 and 400 μg of the antigen conjugate isreproduced.

[0495] ELISAs were done in triplicate for each immunizing peptide andrabbit serum, and performed as follows: 96 well microplates were coatedat 4° C. during 16 hours with either 100 ng of CASB7439 peptide or 100ng of KLH as coating antigens. After 2-hour saturation at 25° C. withBSA (1 mg/ml), serial dilution of the rabbit sera was added for 2 hoursat 25° C. (starting at dilution {fraction (1/100)}). Anti rabbitantibody, conjugated with universal-HRP, was then added at dilution{fraction (1/1000)} for 2 hours at 25° C. as secondary antibody. Plateswere washed and OPD (0.4 mg/ml) is added for 30 minutes at 25° C.Reaction was stopped with H₂SO₄ 4M, and OD measured at 492 nm.

[0496] CASB7439 peptide immunization ELISA read-out results are shown intables 7 to 10 and FIGS. 32 to 35. Results clearly show an antibodyimmune response against distinct CASB7439 synthetic peptide can beraised in several rabbits in a dose-dependant manner. This suggestsCASB7439 candidate antigen is indeed immunogenic and, when properlyformulated with adjuvant as a vaccine, CASB7439 vaccine is able toinduce a strong and specific antibody immune response. TABLE 7 CASB7439peptide 1 immunization of rabbit SB598: ELISA read-outs. CASB7439Peptide PPI (preimmune) PP GP Replicate: Replicate: Replicate: KLHCarrier Dilution 1 2 3 1 2 3 1 2 3 PPI PP GP 100 0.096 0.116 0.123 2.7342.753 2.821 3.305 3.305 3.306 0.137 3.532 3.532 300 0.066 0.076 0.0821.622 1.582 1.535 2.813 2.79 2.85 0.084 3.405 3.445 900 0.056 0.0640.067 0.587 0.582 0.532 1.336 1.221 1.333 0.078 2.899 2.55 2700 0.0590.067 0.059 0.243 0.216 0.183 0.592 0.435 0.472 0.064 1.548 1.246 81000.121 0.057 0.068 0.113 0.099 0.086 0.23 0.163 0.167 0.061 0.623 0.44324300 0.051 0.056 0.052 0.09 0.072 0.065 0.124 0.096 0.094 0.063 0.2690.2 72900 0.055 0.058 0.056 0.061 0.08 0.057 0.07 0.065 0.17 2187000.056 0.052 0.055 0.065 0.056 0.054 0.057 0.059 0.057

[0497] TABLE 8 CASB7439 peptide 1 immunization of rabbit SB599: ELISAread-outs. CASB7439 Peptide PPI (preimmune) PP GP Replicate: Replicate:Replicate: KLH Carrier Dilution 1 2 3 1 2 3 1 2 3 PPI PP GP 100 0.0680.053 0.065 3.172 3.347 3.294 3.354 3.248 3.283 0.105 3.582 3.493 3000.047 0.057 0.06 2.468 2.399 2.437 2.748 2.855 2.848 0.082 3.415 3.391900 0.053 0.057 0.058 1.315 1.19 1.27 1.603 1.45 1.43 0.07 2.941 2.7632700 0.055 0.06 0.059 0.598 0.472 0.493 0.669 0.551 0.528 0.067 1.7091.526 8100 0.054 0.055 0.056 0.207 0.164 0.171 0.214 0.185 0.178 0.0650.625 0.573 24300 0.053 0.054 0.056 0.098 0.087 0.092 0.104 0.091 0.0920.067 0.263 0.249 72900 0.057 0.057 0.059 0.064 0.063 0.061 0.08 0.0670.067 218700 0.065 0.06 0.061 0.057 0.059 0.056 0.056 0.056 0.058

[0498] TABLE 9 CASB7439 peptide 2 immunization of rabbit SB600: ELISAread-outs. CASB7439 Peptide PPI (preimmune) PP GP Replicate: Replicate:Replicate: KLH Carrier Dilution 1 2 3 1 2 3 1 2 3 PPI PP GP 100 0.0780.08 0.074 3.195 3.225 3.179 3.253 3.236 3.256 0.116 3.545 3.573 3000.063 0.073 0.064 2.758 2.823 2.874 3.216 3.229 3.251 0.08 3.479 3.502900 0.068 0.063 0.062 1.606 1.536 1.699 2.585 2.585 2.581 0.081 2.4852.92 2700 0.066 0.064 0.065 0.843 0.746 0.704 1.361 1.44 1.328 0.0741.225 2.026 8100 0.064 0.068 0.064 0.48 0.392 0.401 0.778 0.813 0.7220.068 0.721 1.067 24300 0.063 0.069 0.062 0.21 0.163 0.224 0.283 0.2990.247 0.069 0.237 0.405 72900 0.068 0.067 0.066 0.114 0.101 0.098 0.1310.135 0.115 218700 0.076 0.065 0.063 0.078 0.073 0.071 0.08 0.08 0.082

[0499] TABLE 10 CASB7439 peptide 2 immunization of rabbit SB601: ELISAread-outs. CASB7439 Peptide PPI (preimmune) PP GP Replicate: Replicate:Replicate: KLH Carrier Dilution 1 2 3 1 2 3 1 2 3 PPI PP GP 100 0.0690.072 0.08 2.443 2.536 2.57 2.973 3.019 3.077 0.117 3.497 3.507 3000.064 0.065 0.065 1.427 1.469 1.592 2.366 2.381 2.44 0.077 3.156 3.328900 0.064 0.063 0.065 0.704 0.699 0.751 1.343 1.368 1.314 0.068 2.4162.695 2700 0.062 0.064 0.065 0.302 0.302 0.298 0.76 0.589 0.532 0.0781.106 1.29 8100 0.062 0.065 0.065 0.126 0.323 0.129 0.221 0.218 0.2050.183 0.49 0.484 24300 0.061 0.064 0.06 0.075 0.078 0.079 0.111 0.1 0.090.095 0.181 0.176 72900 0.063 0.065 0.063 0.072 0.071 0.073 0.102 0.080.074 218700 0.064 0.062 0.065 0.069 0.067 0.074 0.069 0.068 0.072

[0500] 17.2.3 Monoclonal Antibodies:

[0501] Immunisation

[0502] 5 BALB/c mice are immunized 3 times at 3 week intervals with 5 μgof purified protein. Bleedings are performed 14 days post II and 1 weekpost 3. The sera are tested by ELISA on purified protein used as coatedantigen. Based on these results (midpoint dilution >10000) one mouse isselected for fusion.

[0503] Fusion/HATselection

[0504] Spleen cells are fused with the SP2/0 myeloma according to astandard protocol using PEG 40% and DMSO 5%. Cells are then seeded in 96well plates 2.5×104-105 cells/well and resistant clones will be selectedin HAT medium. The supernatant of these hybridomas will be tested fortheir content in specific antibodies and when positive, will besubmitted to 2 cycles of limited dilution. After 2 rounds of screening,3 hybridomas will be chosen for ascitis production.

[0505] 17.2.3 Immunohistochemistry

[0506] When antibodies are available, immunostaining is performed onnormal or cancer tissue sections, in order to determine:

[0507] the level of expression of the antigen of the invention in cancerrelative to normal tissue or the proportion of cancer of a certain typeexpressing the antigen

[0508] if other cancer types also express the antigen

[0509] the proportion of cells expressing the antigen in a cancer tissue

[0510] Tissue Sample Preparation

[0511] After dissection, the tissue sample is mounted on a cork disk inOCT compound and rapidly frozen in isopentane previously super cooled inliquid nitrogen (−160° C.). The block will then be conserved at −70° C.until use. 7-10 μm sections will be realised in a cryostat chamber (−20,−30° C.).

[0512] Staining

[0513] Tissue sections are dried for 5 minutes at room temperature(“RT”), fixed in acetone for 10 minutes at RT, dried again, andsaturated with PBS 0.5% BSA 5% serum. After 30 minutes at RT either adirect or indirect staining is performed using antigen specificantibodies. A direct staining leads to a better specificity but a lessintense staining whilst an indirect staining leads to a more intense butless specific staining.

[0514] 17.3 Analysis of Human Cellular Immune Responses to the Antigenof the Invention

[0515] The immunological relevance of the antigen of the invention canbe assessed by in vitro priming of human T cells. All T cell lymphocytelines and dendritic cells are derived from PBMCs (peripheral bloodmononuclear cells) of healthy donors (HLA-A2 subtype). An HLA-A2.1/Kbtransgenic mouse model is also used for screening of HLA-A2.1 peptides.

[0516] CD8+ T-Cell Response

[0517] Newly discovered antigen-specific CD8+ T cell lines are raisedand maintained by weekly in vitro stimulation. The lytic activity andthe γ-IFN production of the CD8+ lines in response to the antigen orantigen derived-peptides is tested using standard procedures.

[0518] Two strategies to raise the CD8+ T cell lines are used: apeptide-based approach and a whole gene-based approach. Both approachesrequire the full-length cDNA of the newly discovered antigen in thecorrect reading frame to be either cloned in an appropriate deliverysystem or to be used to predict the sequence of HLA binding peptides.

[0519] Peptide-Based Approach

[0520] Briefly, transgenic mice are immunized with adjuvanted HLA-A2peptides, those unable to induce a CD8+ response (as defined by anefficient lysis of peptide-pulsed autologous spleen cells) will befurther analyzed in the human system.

[0521] Human dendritic cells (cultured according to Romani et al.) willbe pulsed with peptides and used to stimulate CD8+-sorted T cells (byFacs). After several weekly stimulations, the CD8+ lines will be firsttested on peptide-pulsed autologous BLCL (EBV-B transformed cell lines).To verify the proper in vivo processing of the peptide, the CD8+ lineswill be tested on cDNA-transfected tumour cells (HLA-A2 transfectedLnCaP, Skov3 or CAMA tumour cells).

[0522] Whole Gene-Based Approach

[0523] CD8+ T cell lines will be primed and stimulated with eithergene-gun transfected dendritic cells, retrovirally transducedB7.1-transfected fibroblasts, recombinant pox virus or adenovirusinfected dendritic cells. Virus infected cells are very efficient topresent antigenic peptides since the antigen is expressed at high levelbut can only be used once to avoid the over-growth of viral T cellslines.

[0524] After alternated stimulations, the CD8+ lines are tested oncDNA-transfected tumour cells as indicated above. Peptide specificityand identity is determined to confirm the immunological validation.

[0525] CD4+ T-Cell Response

[0526] Similarly, the CD4+ T-cell immune response can also be assessed.Generation of specific CD4+ T-cells is made using dendritic cells loadedwith recombinant purified protein or peptides to stimulate the T-cells.

[0527] The above description fully discloses how to make and use thepresent invention. However, this invention is not limited to theparticular embodiments described hereinabove, but includes allmodification thereof within the scope of the appended claims and theirequivalents. Those skilled in the art will recognize through routineexperimentation that various changes and modifications can be madewithout departing from the scope of this invention. The variousreferences to journals, patents and other patent applications that arecited herein are incorporated by reference herein as though fully setforth.

[0528] Sequence Information SEQ ID NO: 1GTACCTTGCTTTGGGGGCGCACTAAGTACCTGCCGGGAGCAGGGGGCGCACCGGGAACTCGCAGATTTCGCCAGTTGGGCGCACTGGGGATCTGTGGACTGCGTCCGGGGGATGGGCTAGGGGGACATGCGCACGCTTTGGGCCTTACAGAATGTGATCGCGCGAGGGGGAGGGCGAAGCGTGGCGGGAGGGCGAGGCGAAGGAAGGAGGGCGTGAGAAAGGCGACGGCGGCGGCGCGGAGGAGGGTTATCTATACATTTAAAAACCAGCCGCCTGCGCCGCGCCTGCGGAGACCTGGGAGAGTCCGGCCGCACGCGCGGGACACGAGCGTCCCACGCTCCCTGGCGCGTACGGCCTGCCACCACTAGGCCTCCTATCCCCGGGCTCCAGACGACCTAGGACGCGTGCCCTGGGGAGTTGCCTGGCGGCGCCGTGCCAGAAGCCCCCTTGGGGCGCCACAGTTTTCCCCGTCGCCTCCGGTTCCTCTGCCTGCACCTTCCTGCGGCGCGCCGGGACCTGGAGCGGGCGGGTGGATGCAGGCGCGATGGACGGCGGCACACTGCCCAGGTCCGCGCCCCCTGCGCCCCCCGTCCCTGTCGGCTGCGCTGCCCGGCGGAGACCCGCGTCCCCGGAACTGTTGCGCTGCAGCCGGCGGCGGCGACCGGCCACCGCAGAGACCGGAGGCGGCGCAGCGGCCGTAGCGCGGCGCAATGAGCGCGAGCGCAACCGCGTGAAGCTGGTGAACTTGGGCTTCCAGGCGCTGCGGCAGCACGTGCCGCACGGCGGCGCCAGCAAGAAGCTGAGCAAGGTGGAGACGCTGCGCTCAGCCGTGGAGTACATCCGCGCGCTGCAGCGCCTGCTGGCCGAGCACGACGCCGTGCGCAACGCGCTGGCGGGAGGGCTGAGGCCGCAGGCCGTGCGGCCGTCTGCGCCCCGCGGGCCGCCAGGGACCACCCCGGTCGCCGCCTCGCCCTCCCGCGCTTCTTCGTCCCCGGGCCGCGGGGGCAGCTCGGAGCCCGGCTCCCCGCGTTCCGCCTACTCGTCGGACGACAGCGGCTGCGAAGGCGCGCTGAGTCCTGCGGAGCGCGAGCTACTCGACTTCTCCAGCTGGTTAGGGGGCTACTGAGCGCCCTCGACCTATGAGCCTCAGCCCCGGAAGCCGAGCGAGCGGCCGGCGCGCTCATCGCCGGGGAGCCCGCCAGGTGGACCGGCCCGCGCTCCGCCCCCAGCGAGCCGGGGACCCACCCACCACCCCCCGCACCGCCGACGCCGCCTCGTTCGTCCGGCCCAGCCTGACCAATGCCGCGGTGGAAACGGGCTTGGAGCTGGCCCCATAAGGGCTGGCGGCTTCCTCCGACGCCGCCCCTCCCCACAGCTTCTCGACTGCAGTGGGGCGGGGGGCACCAACACTTGGAGATTTTTCCGGAGGGGAGAGGATTTTCTAAGGGCACAGAGAATCCATTTTCTACACATTAACTTGAGCTGCTGGAGGGACACTGCTGGCAAACGGAGACCTATTTTTGTACAAAGAACCCTTGACCTGGGGCGTAATAAAGATGACCTGGACCCCTGCCCCCACTATCTGGAGTTTTCCATGCTGGCCAAGATCTGGACACGAGCAGTCCCTGAGGGGCGGGGTCCCTGGCGTGAGGCCCCCGTGACAGCCCACCCTGGGGTGGGTTTGTGGGCACTGCTGCTCTGCTAGGGAGAAGCCTGTGTGGGGCACACCTCTTCAAGGGAGCGTGAACTTTATAAATAATCAGTTCTGTTTAAAAAAAAAAAAAAAAAAA SEQ IDNO: 2MDGGTLPRSAPPAPPVPVGCAARRRPASPELLRCSRRRRPATAETGGGAAAVARRNERERNRVKLVNLGFQALRQHVPHGGASKKLSKVETLRSAVEYIRALQRLLAEHDAVRNALAGGLRPQAVRPSAPRGPPGTTPVAASPSRASSSPGRGGSSEPGSPRSAYSSDDSGCEGALSPAERELLDFSSWLGGY SEQ ID NO: 3MSAPAARSASGAEAHRSRALSSPLTSWRSRVARAPQDSARLRSRCRPTSRRNAGSRAPSCPRGPGTKKRGRARRRPGWSLAARGAQTAARPAASALPPARCARRRARPAGAAARGCTPRLSAASPPCSASCWRRRAARAAAAPGSPSSPASRGCARAHCAALRPLRRLRSLRWPVAAAGCSATVPGTRVSAGQRSRQGRGAQGARTWAVCRRPSRLHPPARSRSRRAAGRCRQRNRRRRGKLWRPKGASGTAPPGNSPGHAS SEQ ID NO: 4GTACCTTGCTTTGGGGGCGCACTAAGTACCTGCCGGGAGCAGGGGGCGCACCGGGAACTCGCAGATTTCGCCAGTTGGGCGCACTGGGGATCTGTGGACTGCGTCCGGGGGATGGGCTAGGGGGACATGCGCACGCTTTGGGCCTTACAGAATGTGATCGCGCCGAGGGGGAGGGCCGAAGCGTGGCGGGAGGGCGAGGCGAAGGAAGGAGGGCGTGAGAAAGGCGACGGCGGCGGCGCGGAGGAGGGTTATCTATACATTTAAAAACCAGCCGCCTGCGCCGCGCCTGCGGAGACCTGGGAGAGTCCGGCCGCACGCGCGGGACACGAGCGTCCCACGCTCCCTGGCGCGTACGGCCTGCCACCACTAGGCCTCCTATCCCCGGGCTCCAGACGACCTAGGACGCGTGCCCTGGGGAGTTGCCTGGCGGCGCCGTGCCAGAAGCCCCCTTGGGGCGCCACAGTTTTCCCCGTCGCCTCCCGTTCCTCTGCCTGCACCTTCCTGCGGCGCGCCGGGACCTGGAGCGGGCGGGTGGATGCAGGCGCGATGGACGGCGGCACACTGCCCAGGTCCGCGCCCCCTGCGCCCCCCGTCCCTGTCGGCTGCGCTGCCCGGCGGAGACCCGCGTCCCCGGAACTGTTGCGCTGCAGCCGGCGGCGGCGACCGGCCACCGCAGAGACCGGAGGCGGCGCAGCGGCCGTAGCGCGGCGCAATGAGCGCGAGCGCAACCGCGTGAAGCTGGTGAACTTGGGCTTCCAGGCGCTGCGGCAGCACGTGCCGCACGGCGGCGCCAGCAAGAAGCTGAGCAAGGTGGAGACGCTGCGCTCAGCCGTGGAGTACATCCGCGCGCTGCAGCGCCTGCTGGCCGAGCACGACGCCGTGCGCAACGCGCTGGCGGGAGGGCTGAGGCCGCAGGCCGTGCGGCCGTCTGCGCCCCGCGGGCCGCCAGGGACCACCCCGGTCGCCGCCTCGCCCTCCCCCGCTTCTTCGTCCCCGGGCCGCGGGGGCAGCTCGGAGCCCGGCTCCCCGCGTTCCGCCTACTCGTCGGACGACAGCGGCTGCGAAGGCGCGCTGAGTCCTGCGGAGCGCGAGCTACTCGACTTCTCCAGCTGGTTAGGGGGCTACTGAGCGCCCTCGACCTAATAAGCCTCAAGCCCCGGAAACCCGAGCGAACGGGCCGGCGCGCTTCATCGCCGGGGAAGCCCGCCAAGGTGGACCGGGCCCGCGCTCCGCCCCCAGCGAGCCGGGGACCCACCCACCACCCCCCGCACCGCCGACGCCGCCTCGTTCGTCCGGCCCAGCCTGACCAATGCCGCGGTGGAAACGGGCTTGGAGCTGGCCCCATAAGGGCTGGCGGCTTCCTCCGACGCCGCCCCTCCCCACAGCTTCTCGACTGCAGTGGGGCGGGGGGCACCAACACTTGGAGATTTTTCCGGAGGGGAGAGGATTTTCTAAGGGCACAGAGAATCCATTTTCTACACATTAACTTGAGCTGCTGGAGGGACACTGCTGGCAAACGGAGACCTATTTTTGTACAAAGAACCCTTGACCTGGGGCGTAATAAAGATGACCTGGACCCCTGCCCCCACTATCTGGAGTTTTCCATGCTGGCCAAGATCTGGACACGAGCAGTCCCTGAGGGGCGGGGTCCCTGGCGTGAGGCCCCCGTGACAGCCCACCCTGGGGTGGGTTTGTGGGCACTGCTGCTCTGCTAGCGAGAAGCCTGTGTGGGGCACACCTCTTCAAGGGAGCGTGAACTTTATAAATAAATCAGTTCTGTTTAAAAAAAAAAAAAAAAAAAAAACCGAGGGGGGGCCCGGAGCCAACAAA SEQ ID NO: 5GGTAAACAGAACTGATTTATTTATAAAGTTCACGCTCCCTTGAAGAGGTGTGCCCCACACAGGCTTCTCCCTAGCAGAGCAGCAGTGCCCACAAACCCACCCCAGGGTGGGCTGTCACGGGGGCCTCACGCCAGGGACCCCGCCCCTCAGGGACTGCTCGTGTCCAGATCTTGGCCAGCATGGAAAACTCCAGATAGTGGGGGCAGGGGTCCAGGTCATCTTTATTACGCCCCAGGTCAAGGGTTCTTTGTACAAAAATAGGTCTCCGTTTGCCAGCAGTGTCCCTCCAGCAGCTCAAGTTAATGTGTAGAAAATGGATTCTCTGTGCCCTTAGAAAATCCTCTCCCCTCCGGAAAAATCTCCAAGTGTTGGTGCCCCCCGCCCCACTGCAGTCGAGAAGCTGTGGGGAGGGGCGGCGTCGGAGGAAGCCGCAGCCCATTATGGGGCCAGCTCCAAGCCCGTTTCCACCGCGGCATTGGTCAGGCTGGGCGGACGAACGAGGCGGCGTCGGCGGTGCGGGGGGTGGTGGGTGGGTCCCCCGCTCGCTGGGGGCGGAGCAGCGGGCCGGTCCACCTGGCGGGCTCCCC SEQ ID NO: 6TTTTTTTTTTTTTTTTTTTAAACAGAACTGATTTATTTATAAAGTTCACGCTCCCTTGAAGAGGTGTGCCCCACACAGGCTTCTCCCTAGCAGAGCAGCAGTGCCCACAAACCCACCCCAGGGTGGGCTGTCACGGGGGCCTCACGCCAGGGACCCCGCCCCTCAGGGACTGCTCGTGTCCAGATCTTGGCCAGCATGGAAAACTCCAGATAGTGGGGGCAGGGGTCCAGGTCATCTTTATTACGCCCCAGGTCAAGGGTTCTTTGTACAAAAATAGGTCTCCGTTTGCCAGCAGTGTCCCTCCAGCAGCTCAAGTTAATGTGTAGAAAATGGATTCTCTGTGCCCTTAGAAAATCCTCTCCCCTCCGGAAAAATCTCCAAGTGTTGGTGCCCCCCGCCCCACTGCAGTCGAGAAGCTGTGGGGAGGGGCGGCGTCGGAGGAAGCCGCCAGCCCTTATGGCGCCAGCTCCAAGCCCGTTTCCACCGCGGCATTCGTCAGGCTGGGCCGGACGAACGAGGCGGCGTCGGCGGTGCGGGGGGTGGTGGGTGGGTCCCCGGCTCGCTGGGGGCGGAGCGCGGGCCGGTCCACCTGGCGGGCTCCCCGGCGATGAGCGCGCCGGCCGCTCGCTCGGCTTCCGGGGCTGAGGCTCATAGGTCGAGGGCGCTCAGTAGCCCCCTAACCAGCTGGAGAAGTCGAGTAGCTCGCGCTCCGCAGGACTCAGCGCGCCTTCGCAGCCGCTGTCGTCCGACGAGTAGGCGGAACGCGGGGAGCCGGGCTCCGAGCTGCCCCCGCGGCCCGGGGACGAAGAAGCGCGGGAGGGCGAGGCGGCGACCGGGGTGGTCCCTGGCGCCCCGCGGGGCGCAGACGGCCGCACGGCCTGCGGCCTCAGCCCTCCCGCCAGCGCGTTGCGCACGGCGTCGTGCTCGGCCAGCAGGCGCTGCAGCGCGCGGATGTACTCCACGGCTGAGCGCAGCGTCTCCACCTTGCTCAGCTTCTTGCTGGCGCCGCCGTGCGGCACGTGCTGCCGCAGCGCCTGGAAGCCCAAGTTCACCAGCTTCACGCGGTTGCGCTCGCGCTCATTGCGCCGCGCTACGGCCGCTGCGCCGCCTCCGGTCTCTGCGGTGGCCGGTCGCCGCCGCCGGCTGCAGCGCAACAGTTCCGGGGACGCGGGTCTCCGCCGGGCAGCGCAGCCGACAGGGACGGGGGGCGCAGGGGGCGCGGACCTGGGCAGTGTGCCGCCGTCCATCGCGCCTGCATCCACCCGCCCGCTCCAGGTCCCGGCGCGCCGCAGGAAGGTGCAGGCAGAGGAACCGGAGGCGACGGGGAAAACTGTGGCGCCCCAAGGGGGCTTCTGGCACGGCGCCGCCAGGCAACTCCCCAGGGCACGCGTCCTAGGTCGTCTGGAGCCCGGGGATAGGAGGCCTAGTGGTGGCAGGCCGTACGCGCCAGGGAGCGTGGGACGCTCGTGTCCCGCGCGTGCGGCCGGACTCTCCCAGGTCTCCGCAGGCGCGGCGCAGGCGGCTGGTTTTTAAATGTATAGATAACCCTCCTCCGCGCCGCCGCCGTCGCCTTTCTCACGCCCTCCTTCCTTCGCCTCGCCCTCCCGCCACGCTTCGCCCTCCCCCTCGCGCGATCACATTCTGTAAGGCCCAAAGCGTGCGCATGTCCCCCTAGCCCATCCCCCGGACGCAGTCCACAGATCCCCAGTGCGCCCAACTGGCGAAATCTGCGAGTTCCCGGTGCGCCCCCTGCTCCCGGCAGGTACTTAGTGCGCCCCCAAGGTAC SEQ ID NO: 7MCRKWILCALRKSSPLRKNLQVLVPPAPLQSRSCGEGRRRRKPPALMGPAPSPFPPRHWSGWAGRTRRRRRCGGWWVGPRLAGGGARARSTLAGFPGDEARRPVRSGFRGLRLIRSRALSSPLTSWRSRVAPAPQDSARLRSRCRPTSRRNAGSRAPSCPRGPGTKKRGRARRRPGWSLAARGAQTAARPAASALPPARCARRRARPAGAAARGCTPRLSAASPPCSASCWRRRAARAAAAPGSPSSPASRGCARAHCAALRPLRRLRSLRWPVAAAGCSATVPGTRVSAGQRSRQGRGAQGARTWAVCRRPSRLHPPARSRSRRAAGRCRQRNRRRRGKLWRPKGASGTAPPGNSPGHAS SEQ ID NO: 8ATGGATCCAAACACTGTGTCAAGCTTTCAGGTAGATTGCTTTCTTTGGCATGTCCGCAAACGAGTTGCAGACCAAGAACTAGGTGATGCCCCATTCCTTGATCGGCTTCGCCGAGATCAGAAATCCCTAAGAGGAAGGGGCAGCACCCTCGGTCTGGACATCGAGACAGCCACACGTGCTGGAAAGCAGATAGTGGAGCGGATTCTGAAAGAAGAATCCGATGAGGCACTTAAAATGACCATGGACGGCGGCACACTGCCCAGGTCCGCGCCCCCTGCGCCCCCCGTCCCTGTCGGCTGCGCTGCCCGGCGGAGACCCGCGTCCCCGGAACTGTTGCGCTGCAGCCGGCGGCGGCGACCGGCCACCGCAGAGACCGGAGGCGGCGCAGCGGCCGTAGCGCGGCGCAATGAGCGCGAGCGCAACCGCGTGAAGCTGGTGAACTTGGGCTTCCAGGCGCTGCGGCAGCACGTGCCGCACGGCGGCGCCAGCAAGAAGCTGAGCAAGGTGGAGACGCTGCGCTCAGCCGTGGAGTACATCCGCGCGCTGCAGCGCCTGCTGGCCGAGCACGACGCCGTGCGCAACGCGCTGGCGGGAGGGCTGAGGCCGCAGGCCGTGCGGCCGTCTGCGCCCCGCGGGCCGCCAGGGACCACCCCGGTCGCCGCCTCGCCCTCCCGCGCTTCTTCGTCCCCGGGCCGCGGGGGCAGCTCGGAGCCCGGCTCCCCGCGTTCCGCCTACTCGTCGGACGACAGCGGCTGCGAAGGCGCGCTGAGTCCTGCGGAGCGCGAGCTACTCGACTTCTCCAGCTGGTTAGGGGGCTACACTAGTGGCCACCATCACCATCACCATTAA SEQ ID NO: 9ATGGATCCAAACACTGTGTCAAGCTTTCAGGTAGATTGCTTTCTTTGGCATGTCCGCAAACGAGTTGCAGACCAAGAACTAGGTGATGCCCCATTCCTTGATCGGCTTCGCCGAGATCAGAAATCCCTAAGAGGAAGGGGCAGCACCCTCGGTCTGGACATCGAGACAGCCACACGTGCTGGAAAGCAGATAGTGGAGCGGATTCTGAAAGAAGAATCCGATGAGGCACTTAAAATGACCATGGACGGCGGCACCCTGCCGCGTTCCGCGCCGCCGGCGCCGCCAGTTCCGGTTGGCTGCGCTGCCCGTCGCCGTCCCGCGTCCCCGGAACTGCTGCGCTGCAGCCGTCGCCGTCGCCCGGCCACCGCAGAGACCGGAGGCGGCGCAGCGGCCGTAGCGCGGCGCAATGAGCGCGAGCGCAACCGCGTGAAGCTGGTGAACTTGGGCTTCCAGGCGCTGCGGCAGCACGTGCCGCACGGCGGCGCCAGCAAGAAGCTGAGCAAGGTGGAGACGCTGCGCTCAGCCGTGGAGTACATCCGCGCGCTGCAGCGCCTGCTGGCCGAGCACGACGCCGTGCGCAACGCGCTGGCGGGAGGCTGAGGCCGCAGGCCGTGCGGCCGTCTGCGCCCCGCGGGCCGCCAGGGACCACCCCGGTCGCCGCCTCGCCCTCCCGCGCTTCTTCGTCCCCGGGCCGCGGGGGCAGCTCGGAGCCCCGCTCCCCGCGTTCCGCCTACTCGTCGGACGACAGCGGCTGCGAAGGCGCGCTGAGTCCTGCGGAGCGCGAGCTACTCGACTTCTCCAGCTGGTTAGGGGGCTACACTAGTGGCCACCATCACCATCACCATTAA SEQ ID NO: 10MDPNTVSSFQVDCFLWHVRKRVADQELGDAPFLDRLRRDQKSLRGRGSTLGLDIETATRAGKQIVERILKEESDEALKMTMDGGTLPRSAPPAPPVPVGCAARRRPASPELLRCSRRRRPATAETGGGAAAVARRNERERNRVKLVNLGFQALRQHVPHGGASKKLSKVETLRSAVEYIRALQRLLAEHDAVRNALAGGLRPQAVRPSAPRGPPGTTPVAASPSRASSSPGRGGSSEPGSPRSAYSSDDSGCEGALSPAERELLDFSSWLGGYTSGHHHHHH SEQID NO: 11MYSTAERSVSTLLSFLLAPPCGTCCRSAWKPKFTSFTRLRSRSLRRATAAAPPPVSAVAGRRRRLQRNSSGDAGLRRAAQPTGTGGAGGADLGSVPPSIAPASTRPLQVPARRRKVQAEEPEATGKTVAPQGGFWHGAARQLPRARVLGRLEPGDRRPSGGRPYAPGSVGRSCPARAAGLSQVSAGAAQAAGF SEQ ID NO: 12MEAHLDWYGVPGLQEASDACPRESCSSALPEAREGANVHFPPHPVPREHFSCAAPELVAGAQGLNASLMDGGALPRLMPTSSGVAGACAARRRQASPELLRCSRRRRSGATEASSSSAAVARRNERERNRVKLVNLGFQALRQHVPHGGANKKLSKVETLRSAVEYIRALQRLLAEHDAVRAALAGGLLTPATPPSDECAQPSASPASASLSCASTSPSPDRLGCSEPTSPRSAYSSEESSCEGELSPMEQELLDFSSWLGGY SEQ ID NO: 13GCCCGGAGCATGGAAGCACGTCAGCTAGGCCATGAACTGCACCCGGGAGGGGTGGGGGTGGAAGCGCACGGTGTCAGCTTTGCAGAATGTGTACACCAAGGGGAGGGCGAGGCGAAGGAAGGAGGGCGTAAGAAAGGAGGCGGTGGCGGGGCGGAGGAGATTATCTATACTTTTTAAAAAAAAGGAGCCTCTTAGCCGCGTAAAGGAGACTTGGGGAGCGCCTGACAGCACGCGCGGGACACGAGAGTACCACGCTTCCCTACTCTTTTCAGACCTTGACTGGTACGGGGTCCCAGGACTGCAGGAGGCCAGCGACGCGTGCCCTAGGGAGTCCTGCAGCAGTGCCCTGCCTGAGGCCCGTGAAGGTGCAAACGTCCACTTCCCACCGCACCCGGTTCCTCGCGAGCACTTTTCCTGTGCCGCACCAGAACTCGTAGCAGGGGCCCAGGGGCTGAATGCAAGCTTGATGGACGGCGGCGCGCTGCCCAGACTCATGCCCACCTCGTCTGGAGTCGCTGGAGCCTGCGCTGCTCGGCGGAGACAAGCGTCTCCGGAATTGCTGCGCTGCAGCCGGCGGCGGCGATCTGGAGCAACCGAGGCCAGCAGCAGCTCGGCGTCCGTGGCACGCCGCAATGAGCGCGAGCGCAACCGCGTAAAGCTGGTAAACTTGGGCTTCCAGGCGCTGCGGCAGCACGTGCCGCACGGCGGCGCCAACAAGAAGCTGAGTAAGGTGGAGACGCTGCGCTCCGCGGTAGAGTACATTCGTGCGCTGCAGCGGCTGCTCGCAGAGCACGACACGGTGCGGCCGGNGCTCGCTGGGGGGCTGTTAACACCCGCTACTCCGCCGTCCGATGAGTGCACGCAGCCCTCTGCCTCCCCTGCCAGCGGGTCTCTGTCCTGCGCCTCTACGTCTCCGTCCCGGACCCTGGGCTGCTCTGAGCCTACCTCCCCGCGCTCCGCCTACTCGTCGGAGGAAAGCAGCTGCGAGGGAGAGCTAAGCCCGATGGAGCAGGAGCTGCTTGACTTTTCCAGTTGGTTAGGGGGCTACTGA SEQ ID NO: 14MESHFNWYGVPRLQKASDACPRESCSSALPEAREGANVHFPPHPVPREHFSCGAPKPVAGAPALNASLMDGGALPRLVPTSSGVAGACTARRRPPSPELLRCSRRRRSGATEASSSSAAVARRNERERNRVKLVNLGFQALRQHVPHGGANKKLSKVETLRSAVEYIRALQRLLAEHDAVRAALSGGLLTPATRPSDVCTQPSASPASASLSCTSTSPDRLGCSEPASPRSAYSSEDSSCEGETYPMGQMFDFSNWLGGY SEQ ID NO:15TTCACCCGGCTGCAAGCGCTAGGTGTACGGAGACCTGGCAGCTCTTGGGGCTTAAGGACTGAGCRCCAGAGCCGGTGGAGGTTCCTGTGGAGTACATTCGGACCCTCTCACAGCCCCCGAGAGTGCGGGACGTGCGGAGCGCAGTTCGGGATCTGCACTCGAGGACTTGTCGAGGACGCATTAAGCTAAGCATCTGCTCGGAGCATGGAATCGCACTTTAACTGGTACGGGGTCCCAAGGCTCCAGAAGGCTAGCGACGCGTGCCCTAGGGAATCCTGCAGCAGTGCCCTGCCTGAGGCCCGTGAAGGTGCGAACGTCCACTTCCCACCGCACCCGGTTCCTCGCGAGCACTTTTCCTGTGGCGCACCGAAACCCGTAGCGGGGGCCCCGGCGCTGAATGCAAGCTTGATGGACGGCGGCGCGCTGCCCAGACTCGTGCCCACCTCGTCTGGAGTCGCTGGAGCCTGCACTGCTCGGCGGAGACCCCCGTCCCCGGAACTGCTTCGCTGCAGCCGACGGCGGCGATCGGGAGCAACCGAGGCCAGCAGCAGCTCGGCGGCCGTGGCACGCCGCAATGAGCGTGAGCGCAACCGCGTAAAGCTGGTAAACTTGGGCTTCCAGGCGCTGCGGCAGCACGTGCCGCACGGCGGCGCCAACAAGAAGCTGAGTAAGGTGGAGACGCTGCGCTCCGCGGTAGAGTACATCCGTGCGCTGCAGCGGCTGCTAGCAGAGCACGACGCGGTGCGTGCTGCGCTCTCTGGGGGTCTATTAACACCCGCTACTCGGCCGTCCGATGTGTGCACGCAGCCCTCCGCCTCCCCTGCCAGCGCGTCTCTGTCCTGCACCTCTACATCCCCAGACCGCCTAGGCTGCTCCGAGCCTGCCTCTCCGCGCTCCGCCTACTCGTCGGAGGACAGCAGCTGCGAGGGAGAGACTTACCCGATGGGGCAGATGTTTGACTTTTCCAATTGGTTAGGGGGCTACTGAGCACCCCACACCCCTAAGCTGCGTCCCTGGGTGTCCCCTGGTGGACCTACCTGCGTTTCTTGCCCAGGAAACCTGGGCCCATGCCTTACCCATGCTGTCTAGTGCAGCCTGACCAAATGCCAAGTACTGACCTCTGCTCGGCCTCCACGCCGCGGAATGACATCTTCCATCTCCCAGTCCTTGCCGAACCAGGACTTGGAAATTTCTCAGGAGAAAGAATTTTACAATGACAATCTGCTTTTTATCAATTAACTTGAACTGCTGGAGGACTCTGCTGAAAATATGAAGAATTATTTTTATACAAAGGATCCTTAAGCTTGGAGCACAATAAAGATGACCTCTGTCTCTCACCCCCACTGTCTAGAACTTTCCAACCTGGCCAAAGTGTGGACGGGTCGGGCCCTGAGGGCAAGATGCCTGGCTGCACCCTTCTTCCTCTTCCGAAGCCTATCCTGACGCTGATGTTTGGCCAGTGTGGGAACCCTGCTATTGCAAAGTGTACTATTCTATAAAAGTTGTTTTTCATTGGAAAGGAATTC SEQ ID NO: 16 KLVNLGFQAL SEQ ID NO: 17 ELLDFSSWL SEQ IDNO: 18 RLLAEHDAV SEQ ID NO: 19 KLVNLGFQA SEQ ID NO: 20 EYIRALQRL SEQ IDNO: 21 EYIRALQRLL SEQ ID NO: 22 AVRNALAGGL SEQ ID NO: 23 SEPGSPRSAY SEQID NO: 24 VETLRSAVEY SEQ ID NO: 25 IRALQRLLA SEQ ID NO: 26 LRPQAVRPS SEQID NO: 27 LRQHVPHGG SEQ ID NO: 28 LGFQALRQH SEQ ID NO: 29 VRNALAGGL SEQID NO: 30 YIRALQRLL SEQ ID NO: 31 LVNLGFQAL SEQ ID NO: 32 VEYIRALQR SEQID NO: 33 LLRCSRRRR SEQ ID NO: 34ATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCCCATGGACGGCGGCACACTGCCCAGGTCCGCGCCCCCTGCGCCCCCCGTCCCTGTCGGCTGCGCTGCCCGGCGGAGACCCGCGTCCCCGGAACTGTTGCGCTGCAGCCGGCGGCGGCGACCGGCCACCGCAGAGACCGGAGGCGGCGCAGCGGCCGTAGCGCGGCGCAATGAGCGCGAGCGCAACCGCGTGAAGCTGGTGAACTTGGGCTTCCAGGCGCTGCGGCAGCACGTGCCGCACGGCGGCGCCAGCAAGAAGCTGAGCAAGGTGGAGACGCTGCGCTCAGCCGTGGAGTACATCCGCGCGCTGCAGCGCCTGCTGGCCGAGCACGACGCCGTGCGCAACGCGCTGGCGGGAGGGCTGAGGCCGCAGGCCGTGCGGCCGTCTCCGCCCCGCGGGCCGCCAGGCACCACCCCGGTCGCCGCCTCGCCCTCCCGCGCTTCTTCGTCCCCGGGCCGCGGGGGCAGCTCGGAGCCCGGCTCCCCGCGTTCCGCCTACTCGTCGGACGACAGCGGCTGCGAAGGCGCGCTGAGTCCTGCGGAGCGCGAGCTACTCGACTTCTCCAGCTGGTTAGGGGGCTACACTAGTCTCGAGCACCACCACCACCACCACTGA SEQ ID NO: 35MASMTGGQQMGRDPMDGGTLPRSAPPAPPVPVGCAARRRPASPELLRCSRRRRPATAETGGGAAAVARRNERERNRVKLVNLGFQALRQHVPHGGASKKLSKVETLRSAVEYIRALQRLLAEHDAVRNALAGGLRPQAVRPSAPRGPPGTTPVAASPSRASSSPGRGGSSEPGSPRSAYSSDDSGCEGALSPAERELLDFSSWLGGYTSLEHHHHHH SEQ ID NO: 36ATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCCCATGGACGGTGGTACCCTGCCGCGTTCCGCTCCGCCGGCTCCGCCGGTTCCGGTTGGTTGCGCTGCTCGTCGTCGTCCGGCTTCCCCGGAACTGCTGCGTTGCTCCCGTCGTCGTCGTCCGGCTACCGCAGAGACCGGAGGCGGCGCAGCGGCCGTAGCGCGGCGCAATGAGCGCGAGCGCAACCGCGTGAAGCTGGTGAACTTGGGCTTCCAGGCGCTGCGGCAGCACGTGCCGCACGGCGGCGCCAGCAAGAAGCTGAGCAAGGTGGAGACGCTGCGCTCAGCCGTGGAGTACATCCGCGCGCTGCAGCGCCTGCTGGCCGAGCACGACGCCGTGCGCAACGCGCTGGCGGGAGGGCTGAGGCCGCAGGCCGTGCGGCCGTCTGCGCCCCGCGGGCCGCCAGGGACCACCCCGGTCGCCGCCTCGCCCTCCCGCGCTTCTTCGTCCCCGGGCCGCGGGGGCAGCTCGGAGCCCGGCTCCCCGCGTTCCGCCTACTCGTCGGACGACAGCGGCTGCGAAGGCGCGCTGAGTCCTGCGGAGCGCGAGCTACTCGACTTCTCCAGCTGGTTAGGGGGCTACACTAGTCTCGAGCACCACCACCACCACCACTGA SEQ ID NO:37ATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCCCATGGATGGTGGTACTCTGCCACGTTCTGCTCCGCCAGCTCCACCGGTTCCGGTAGGTTGTGCTGCACGTCGCCGTCCAGCTTCTCCAGAACTGCTTCGTTGTTCTCGTCGCAGACGTCCAGCTACCGCAGAGACCGGAGGCGGCGCAGCGGCCGTAGCGCGGCGCAATGAGCGCGAGCGCAACCGCGTGAAGCTGGTGAACTTGGGCTTCCAGGCGCTGCGGCAGCACGTGCCGCACGGCGGCGCCAGCAAGAAGCTGAGCAAGGTGGAGACGCTGCGCTCAGCCGTGGAGTACATCCGCGCGCTGCAGCGCCTGCTGGCCGAGCACGACGCCGTGCGCAACGCGCTGGCGGGAGGGCTGAGGCCGCAGGCCGTGCGGCCGTCTGCGCCCCGCGGGCCGCCAGGGACCACCCCGGTCGCCGCCTCGCCCTCCCGCGCTTCTTCGTCCCCGGGCCGCGGGGGCAGCTCGGAGCCCGGCTCCCCGCGTTCCGCCTACTCGTCGGACGACAGCGGCTGCGAAGGCGCGCTGAGTCCTGCGGAGCGCGAGCTACTCGACTTCTCCAGCTGGTTAGGGGGCTACACTAGTCTCGAGCACCACCACCACCACCACTGA

What is claimed:
 1. A method of treating or preventing cancer in apatient comprising the steps of administering a therapeuticallyeffective amount of a polypeptide comprising an amino acid sequencehaving at least 70% sequence identity to the amino acid sequence setforth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12 or SEQ ID NO:14 over the entire length of SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12or SEQ ID NO:14 respectively, wherein the polypeptide may optionallycomprise a fusion partner or an affinity tag, wherein administration ofsaid polypeptide to said patient induces an immune response to a tumourantigen.
 2. The method of claim 1, further comprising admixing thepolypeptide with an adjuvant.
 3. The method of claim 1, wherein thetumour antigen comprises CASB7439.
 4. The method of claim 1, wherein thepatient has or has a potential to contract a cancer comprisingcolorectal, breast or lung cancer.
 5. The method of claim 1, wherein thepolypeptide has at least 95% sequence identity to SEQ ID NO:2.
 6. Amethod of inducing an immunoresponse to CASB7439 in a human or non-humananimal comprising administering a peptide fragment of SEQ ID NO:2 to thehuman or non-human animal.
 7. The method of claim 6, wherein the peptidefragment is selected from the group consisting of SEQ ID NO: 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, and
 33. 8. Themethod of claim 6, wherein the peptide fragment further comprises afusion partner.
 9. The method of claim 6 or 8, further comprisingadmixing the peptide fragment with an adjuvant.
 10. A method ofmanufacturing a medicament for immunotherapeutically treating a patientsuffering from or susceptible to cancer comprising expressing a proteinin a cell comprising a polynucleotide comprising a nucleotide sequencewhich has at least 70% sequence identity to the nucleotide sequence setforth in SEQ ID NO:1 over the entire length of SEQ ID NO:1.
 11. Themethod according to claim 10, wherein the polynucleotide has at least95% sequence identity to SEQ ID NO:1.
 12. The method according to claim10, wherein the patient is suffering from a cancer comprisingcolorectal, breast or lung cancer.
 13. The method according to claim 10,wherein the polynucleotide is selected from the group consisting of (a)a polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:2;(b) the coding region of polynucleotide SEQ ID NO:1; and (c) apolynucleotide obtainable by screening an appropriate library understingent hybridization conditions with a labeled probe having thesequence of SEQ ID NO:1 or a fragment thereof, wherein saidpolynucleotide encodes a polypeptide having simlar properties to thosefo SEQ ID NO:2.
 14. A method of manufacturing a medicament comprising apolypeptide which has at least 70% sequence identity to the amino acidsequence set forth in SEQ ID NO:2 over the entire length of SEQ ID NO:2for the manufacture of a medicament for immunotherapeutically treating apatient suffering from or susceptible to cancer.
 15. The methodaccording to claim 14, wherein the polypeptide has at least 95% sequenceidentity to SEQ ID NO:2.
 16. The method according to claim 14, whereinthe patient is suffering from a cancer comprising colorectal, breast orlung cancer.
 17. An immunogenic fragment of CASB7439, wherein theimmunogenic fragment is immunologically reactive with an antibody thatbinds to and/or a T-cell that reacts with or binds to a polypeptidecomprising SEQ ID NO:2.
 18. A pharmaceutical composition comprising theimmunogenic fragment of claim
 17. 19. A polypeptide comprising the aminoacid sequence set forth in SEQ ID NO:35.
 20. An isolated polynucleotideencoding the polypeptide of claim
 19. 21. An expression vectorcomprising the polynucleotide of claim
 20. 22. A host cell comprisingthe expression vector of claim 21.